CN105018380B - One bacillus amyloliquefaciens BR25 and its cultural method and application - Google Patents
One bacillus amyloliquefaciens BR25 and its cultural method and application Download PDFInfo
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Abstract
The invention discloses a bacillus amyloliquefaciens and its cultural method and applications, belong to biological control plant disease field.The bacterial strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BR25, and deposit number is CGMCC No.10962.The invention also discloses the molecular biological characteristics of above-mentioned bacterial strains and physio-biochemical characteristics, cultural method, and the application in preventing anthracnose of rubber trees.
Description
Technical field
The invention belongs to biological control plant disease fields, are related to a bacillus amyloliquefaciens BR25 (Bacillus
) and its application in preventing anthracnose of rubber trees amyloliquefaciens.
Background technology
Anthracnose of rubber trees is a kind of Common Diseases caused by anthrax-bacilus, it can make rubber leaf fall off, when serious even
Whole strain may be caused dead, be one of three big leaf diseases of rubber tree.It is mainly at present application for the prevention of the disease
Medicine of learning to farm is prevented, and is made a big impact to environment.Then, the method for exploring biological control becomes urgent.Anthrax-bacilus can
It infects various plants and causes anthracnose, such disease distribution is extensive, and harm is serious, is the object of phytopathologist's primary study
One of, anthracnose of rubber trees is wherein to endanger one kind the most serious.Anthracnose of rubber trees is to be infected to cause by colletotrichum gloeosporioides Penz
, it causes countless rubber tree leaf abscissions in Hainan, Yunnan Province or even whole strain is dead, to local Rubber Industry
Cause heavy losses.
Anthrax-bacilus is mainly overwintering in sick branch in the form of mycelium, and generation conidium, which is used as, under the suitable conditions just invades
Dye source.Rubber tree seedling, earsh are until opening tree of tapping rubber at age can be infected by anthrax-bacilus and be fallen ill.Germ can infect tender
The positions such as leaf, petiole, tender tip and fruit cause tender leaf to fall off, the withered and fruit rot of tender tip time, or even form mummy and be suspended on tree
On, the repetition fallen leaves of gum and tender tip can be caused to return when serious withered, postponement, which is opened, cuts the time.The disease is considered as causing natural rubber
One of the main reason for gum underproduction.At present for the prevention of rubber anthracnose still based on chemical bactericide, however, chemical agriculture
Germ drug resistance, toxicity and problem of environmental pollution become increasingly conspicuous caused by medicine, become the main barrier for restricting agricultural sustainable development
Hinder, Biocontrol microorganism has the advantages that environment compatibility is good, nontoxic, is the ideal substitute of chemical pesticide.Therefore, with life
Preventing microorganism prevents anthracnose of rubber trees to realizing that the sustainable development of agricultural is of great significance.
Important component of the biological control as control of plant disease, it is considered to be most development potentiality is important anti-
Control one of method.With the reinforcement of people's environmental consciousness, biological control is met with it to environment, ecology, the safety of human health,
Free from environmental pollution, the features such as of low cost become recent domestic comprehensive control of disease and research hotspot.In recent years, rubber tree
Increasingly severe trend is presented in the occurrence injury of anthracnose.Anthracnose of rubber trees screening of biocontrol agents is ground it can be seen that carrying out
It is a nowadays urgent task in rubber tree production process to study carefully.
Separation screening obtains the biocontrol bacterial strain for having antagonism to anthracnose of rubber trees bacterium out of rubber plant, to rubber
The prevention of gum anthracnose has great importance, but presently relevant technology is still very weak or even blank, so far effectively prevention
The antagonistic bacterium of anthracnose of rubber trees has not been reported, also without the report for the biological pesticide for preventing anthracnose of rubber trees.The present invention
Purpose for the prior art blank provide a bacillus amyloliquefaciens BR25 (Bacillus
) and its application in preventing anthracnose of rubber trees amyloliquefaciens.
Invention content
The purpose of the present invention provides a bacillus amyloliquefaciens BR25 (Bacillus for the blank of the prior art
) and its application in preventing anthracnose of rubber trees amyloliquefaciens.
The purpose of the present invention is achieved by following technical proposals.
The bacterium that the present invention uses is bacillus amyloliquefaciens BR25 bacterial strains, and Classification And Nomenclature is bacillus amyloliquefaciens
Bacillus amyloliquefaciens were preserved in Chinese microorganism strain preservation conservator on 06 05th, 2015
Meeting common micro-organisms center (CGMCC), address：No. 3 Chinese Academy of Sciences microorganisms of city of BeiJing, China Chaoyang District North Star West Road 1 institute
Research institute, postcode 100101；Deposit number is CGMCC NO.10962.
The production bacterial strain BR25 of the present invention is isolated from the rubber plant leaf in Hainan Province Danzhou City treasured island Village, through bacterium colony with
The microexamination of form, staining reaction, customary physiological biochemical characteristic is tested and 16s rDNA sequence analyses, the bacterial strain are accredited as
One new strains of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) have following characteristics：1. bacterial strain is in
Direct rod shape has nose circle or square end often with pairs of or catenation；Gemma ellipse, oval, column, circle, no pod membrane；Gram
Stained positive；2. being referred to by Physiology and biochemistries such as methyl red experiment, tyrosine catabolic experiment, V-P experiments, citrate growth experiments
Mark, and combine《The outstanding Bacteria Identification handbook of uncle》, qualification result BR25 belongs to bacillus subtilis Pseudomonas, as a result with Molecular Identification one
It causes.
The minimal medium of bacillus amyloliquefaciens BR25 is LB culture mediums or YEP culture mediums.
Bacillus amyloliquefaciens BR25's has using carbon source：Sorbierite, rhamnose, vitamin C, threonine, gala
Sugar, proline, mannitol, maltose, cystine, soluble starch, glucose, tyrosine, fructose.
The present invention is investigated bacterial strain BR25 to the sensibility of different antibiotic, lays the first stone for chemical prevention.
The bacterial strain BR25 of the present invention is inhibited to khuskhus anthrax-bacilus, Oak Tree anthrax-bacilus, pepper Fusarium wilt.
The bacterial strain BR25 or its zymotic fluid of the present invention can be used as biological control agent, for preventing plant mycosis
Evil.
The present invention also provides the application of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BR25 a kind of,
The bacterial strain can be used for preventing anthracnose of rubber trees, column flower anthracnose and pepper Fusarium wilt.
The present invention also provides a kind of biological control methods of fungal diseases of plants, to the plant application with fungal disease
Bacterial strain BR25 or its zymotic fluid can also apply the biological control agent containing above-mentioned bacterial strains or its zymotic fluid, can also be with it
Allogene prevents agents application.
The bacterial strain BR25 of the present invention has broad spectrum antibacterial, and culture is simple, adaptable, and antibacterial ability is stablized, Ke Yiguang
The general prevention applied to various plants fungal disease.
Description of the drawings
Fig. 1 is the 16S rDNA PCR product electrophoretograms of BR25 and other biocontrol microorganisms, and swimming lane M is DNA marker, and 4 are
BR25,5 be BR23, and 6 be BR24, and 7 be BR26.
Fig. 2 is the Gram's staining result of bacterial strain BR25.
Fig. 3 is the antagonistic effect figure in bacterial strain BR25 1-10 generations.
Fig. 4 is inhibiting effect of the bacterial strain BR25 to rubber tree anthrax-bacilus.
Fig. 5 is inhibiting effect of the bacterial strain BR25 to pepper droop sickle-like bacteria.
Specific implementation mode
Embodiment 1
Bacterial strain BR25's is separately cultured and screens purifying.
1.1 sampling
Sample is the rubber plant leaf in Hainan Province Danzhou City treasured island Village, and the blade being collected is put into polybag simultaneously
Record.
The disinfection of 1.2 material surfaces is detached with antagonistic bacterium
1.2.1 clear water rinses and clip disease leaf
The dust gravel on blade is washed away with clear water, is dried.It is 5 × 5mm's to be good for intersection clip size in disease with scissors
Vanelets are spare.
1.2.2 sample sterilizes
The minor illness leaf of clip is put into beaker, is impregnated 30 seconds with mercuric chloride, it should be noted that constantly gently stirring during immersion
Then three times with aseptic water washing dynamic minor illness leaf dries, spare.
1.3 bacteriums are separately cultured
The vanelets of clip are placed on PDA solid mediums with sterilized tweezers, 4 pieces are uniformly put per ware, then
Culture dish is sealed with preservative film.It is inverted into constant incubator, 28 DEG C of cultures.It will be grown after 12h carries out observation 48h
Thalline be forwarded on identical culture medium, 28 DEG C culture 48h after purified；Meanwhile the bacterium of gained after purification being used
Gradient dilution is coated in LB plating mediums, and picking single bacterium colony is connected in corresponding medium slant after 28 DEG C of culture 48h.
The screening of 1.4 antagonistic bacteriums
1.4.1 the purifying of bacterium
Infinite dilution method：The slant tube being positioned in 8~10 DEG C of incubators is taken out, in aseptic operating platform, by liquid
Body LB culture mediums are dispensed into conical flask, then choose a small amount of bacterium in conical flask with transfer needle, sealing.It is positioned over 28 DEG C
Shake culture about 12 hours on shaking table are taken out.Then 200 μ L are drawn on LB solid mediums with pipettor, paint daubs is smeared
Uniformly, in 28 DEG C of incubator cultures.Then it is handled again by infinite dilution method and obtains single bacterial strain.
Or using scribing line method of purification：
If the bacterium colony very little to be chosen, and grow slow again, bacterium colony all adheres on oese, and when scribing line can suitably increase
The width and scribing line number of second gradient line can not draw away 4 gradients because when bacterium is few, and the second gradient can be isolated preferably
Single bacterium colony.If the bacterium colony to be chosen is very big, and grows fast again, it is less to choose bacterium, first gradient line want more strokes it is several under, and take up an area
Want small.Second gradient line is drawn less and closeer, and the third and fourth gradient line is then drawn much and dredged, because these bacterium are mainly by the
Three and 4th gradient line detach single bacterium colony.Second line of gradient 2~3 is connect with first gradient line, third and the second gradient line
It is 2~3 line connections.Oese calcination is cooled down with before 3rd gradient scribing line second, can burn to death be sticked on oese in this way
Bacterium.
Embodiment 2
The identification of bacterial strain BR25
1, Microbiological Characteristics：
Morphological feature observation is carried out to the biocontrol bacterial strain for cultivation of crossing in 37 DEG C of incubators, it is found that biocontrol bacterial strain BR25 is in
Direct rod shape, less chaining have nose circle or square end；Gemma ellipse, oval, column, circle, no pod membrane are bacillus.Through leather
Thalline shows bluish violet after blue Albert'stain Albert, is gram-positive bacteria.
2, molecular biology identification：
(1) DNA is extracted：The extraction of genomic DNA is carried out using kit generally in the art；
(2) PCR reacts：Bacterial universal primers 8F and 1492R, by Invitrogen (Shanghai), trading company synthesizes, and structure is such as
Under：8F 5'-AGAGTTTGATCCTGGCTCAG-3'
1492R 5'-GGTTACCTTGTTACGACTT-3',
The results are shown in Figure 1 for the 16S rDNA gene magnifications of biocontrol bacterial strain BR25, amplified band length about 1500bp, with
It is expected that in the same size；
(3) PCR is identified that correct recombinant bacterium send Invitrogen (Shanghai) trading company gene sequencing.
By being compared on the net in NCBI, the homology of as a result display and bacillus subtilis Pseudomonas is 99%, because
This infers that BR25 bacterial strains belong to bacillus subtilis Pseudomonas.
3, the Physiology and biochemistry identification of biocontrol microorganisms
(1) indole test
1. culture medium is prepared：1% tryptone aqueous solution is taken, pH 7.2-7.6 is adjusted, is sub-packed in test tube, is highly test tube
1/3-1/4,121 DEG C sterilizing 20min；
2. after culture medium cooling, 10 μ L strains tested fresh seeds liquid is taken to be inoculated in tryptone fluid nutrient medium；
3. 28 DEG C of cultures, observe result after 2d, 4d；
4. every time when observation, the reagent for being slowly added into 3-5mm high along tube wall occurs red in culture solution surface at liquid layer interface
Color, as positive reaction.If color unobvious, ether about 1mL can be added in culture solution, fully shaking stands a moment, waits for second
There is red, as positive reaction floating to indole reagent is added after liquid level again, in ether and reagent solution bed boundary in ether.
As a result equal redfree generates, and the results are shown in Table 1, therefore it is physiological and biochemical index to analyze biocontrol microorganisms in indoles
For feminine gender.
1 indole test result of table
Note："+" is the positive, and "-" is feminine gender.
(2) citrate growth test
1. culture medium is prepared：NaCl 1.0g, MgSO are taken respectively4·7H2O 0.2g, NH4H2PO40.5g, sodium citrate
2.0g, agar 18.0g, 0.04% phenol red aqueous solution 10mL；Distilled water is supplemented to 1000.0mL, and it is 7.0 to adjust pH, is then added and refers to
Show agent, dispenses test tube；
2. 121 DEG C of sterilizing 20min, put inclined-plane；
3. strains tested single bacterium colony streak inoculation on inclined-plane of picking fresh cultured, 28 DEG C of culture 3-7d；
4. observation experiment result：Culture medium becomes blue or pink as the positive, is otherwise feminine gender.
In addition to the fungi-proofing BR23 that delivers a child is non-discolouring, connecing other biocontrol microorganisms becomes pink, remaining biocontrol bacterial strain is the positive
As shown in table 2；
2 citrate growth test result of table
Note："+" is the positive, and "-" is feminine gender.
(3) most suitable utilization of carbon source measures
1. choose soluble starch, cystine, ascorbic acid, proline, arginine, maltose, tyrosine, mannitol,
Glucose, sorbierite, glycine, rhamnose, fructose, oxalic acid, threonine, galactolipin, etc. utilization of carbon source measure；
2. minimal medium：(NH4)2SO42.0g, NaH2PO4·H2O 0.5g, K2HPO40.5g, MgSO4·7H2O
0.2g, CaCl2·2H2O 0.1g, distilled water are supplemented to 1000.0mL, and pH 6.5 is sub-packed in test tube (every test tube 4mL),
121 DEG C of sterilizing 20min；
3. gained carbon source is configured to 5% aqueous solution, after sterilizing be added test tube in, make its final concentration of 1%；
4. 10 μ L of strains tested seed liquor is taken to be added separately in different carbon source, and blank control is set, then at 28 DEG C,
Under the conditions of 200r/min, training 40h is shaken；
If 5. there is apparent muddiness, illustrate that the carbon source can be utilized；If nothing, needs to be taken out 10 μ L, be transferred to phase
In the new carbon source test tube answered, under similarity condition, continuous culture 3 times；If still without muddiness, prove that this carbon source is not suitable for being supplied
Bacterial strain is tried to utilize.
Experimental result is as follows：
Have using carbon source：Sorbierite, rhamnose, vitamin C, threonine, galactolipin, proline, mannitol, malt
Sugar, cystine, soluble starch, glucose, tyrosine, fructose；
Do not have using carbon source：Arginine, oxalic acid, glycine.
(4) catalase test
1. shaking cultivation strain 16h with fluid nutrient medium；
2. oese dips the fresh bacterium solution of culture 16h, it is coated in and has dripped on the slide for having 3% aqueous hydrogen peroxide solution；
3. observing result：There is bubble to be produced as the positive, bubble-free is feminine gender.
As a result, it has been found that there is bubble generation；Show that catalase experimental result is the positive.
3 catalase test result of table
Note："+" is the positive, and "-" is feminine gender.
(5) oxidase test
1. shaking cultivation strain 16h with LB liquid medium；
2. putting a filter paper in clean culture dish, 1% dimethyl is added dropwise to penylene diamine hydrochloride aqueous solution, only makes filter
Paper moistens, can not overly moist；
3. taking the fresh bacterium solution of culture 16h with oese, it is applied on the filter paper of moistening；
4. observing result：It is the positive that the lawn smeared in 10s, which shows red person, and it is deferred reaction that 10-60s, which shows red person,
60s or more shows red person and disregards, by negative processing.
As a result it shows：The variation of color is not generated in the 60S after smearing lawn；All display is negative.
4 oxidase test result of table
Note："+" is the positive, and "-" is feminine gender.
(6) to the Study of Sensitivity of antibiotic
Bacillus subtilis BR25 is measured to the sensibility without antibiotic with filter paper enzyme, and diameter 7 is prepared with card punch
The filter paper of millimeter, high-temperature sterilization.1 milli is added in the LB solid mediums for dissolving postcooling to 50 degrees Celsius~55 degrees Celsius
Strains tested seed liquor is risen, plate is down flat after shaking up, the filter paper after sterilizing is placed in tablet, antibiosis is then added on filter paper
Plain solution, it is mould that additive amount is equivalent to 50 micrograms per millilitres of ampicillin Amp, 50 micrograms per millilitres of kanamycins Kana, chlorine
50 micrograms per millilitres of plain Chl, 50 micrograms per millilitres of streptomysin Str, 50 micrograms per millilitres of tetracycline Tet, Erythromycin E ry 50
Micrograms per millilitre, 50 micrograms per millilitres of rifampin Rif, 50 micrograms per millilitres of cephamycin C ef.It is positioned over 28 degrees Celsius of constant temperature
Culture in incubator, respectively at 24 hours, 48 hours observation results.As a result as follows：
5 antibiotic resistance experimental result of table
Note："+" is inhibition zone occur, and "-" is inhibition zone do not occur.
Embodiment 3
To the inhibition of plant pathogenic fungi
In the PDA plate center got ready in advance, be inoculated with a diameter of 6mm for trying pathogen fungus block, while away from PDA plate
Tested bacteria is inoculated at symmetrical 2 point of center 25mm, every plant of bacterium is repeated 3 times, and 28 DEG C of constant temperature are inverted culture, are observed after 6d antibacterial
Situation.
Inhibition of the table 5 to disease fungus
Embodiment 4
Fungistatic effects of the bacillus amyloliquefaciens BR25 to pathogen
Culture medium：Tryptone 10.0g, yeast extract 5.0g, sodium chloride 5.0g, agar powder 20g, distilled water are supplemented to
1000.0mL, 121 DEG C of sterilizing 20min.
By carrying out opposite culture experiment to biocontrol microorganisms BR25, as a result show that the 1-10 generations of BR25 stablize antagonism column flower
Careless anthrax-bacilus.Also demonstrate inhibiting effect of the BR25 to Oak Tree anthrax-bacilus and pepper Fusarium wilt.
Described above is only presently preferred embodiments of the present invention, and the interest field of the present invention cannot be limited with this, because
This equivalent changes made in accordance with the claims of the present invention still falls within the range that the present invention is covered.
Claims (5)
1. a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BR25, the preserving number of the bacterial strain are：
CGMCC No.10962。
2. purposes of the bacterial strain BR25 described in claim 1 in preventing fungal diseases of plants；The fungal diseases of plants is by rubber
One or more of gum anthrax bacteria, Character Research on Anthracnose of Stylosanthes spp, pepper droop sickle-like bacteria cause.
3. a kind of biological control agent contains bacterial strain described in claim 1 or its zymotic fluid.
4. purposes of the biological control agent in preventing fungal diseases of plants described in claim 3；The fungal diseases of plants
Caused by one or more of rubber anthrax bacteria, Character Research on Anthracnose of Stylosanthes spp, pepper droop sickle-like bacteria.
5. a kind of biological control method of fungal diseases of plants, which is characterized in that apply right to the plant with fungal disease
It is required that 1 bacterial strain or the biological control agent of claim 3；The fungal disease is by rubber anthrax bacteria, khuskhus anthracnose
One or more of bacterium, pepper droop sickle-like bacteria cause.
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| CN108690821B (en) * | 2017-12-07 | 2019-03-22 | 中国热带农业科学院环境与植物保护研究所 | One plant height imitates bacillus amyloliquefaciens and its microbial inoculum and application |
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| WO2005059112A1 (en) * | 2003-12-17 | 2005-06-30 | Kt & G Co., Ltd | The novel bacillus amyloliquefaciens ktgb0202 and control method of plant pathogenic funzi using that |
| CN102286527A (en) * | 2011-08-30 | 2011-12-21 | 南京农业大学 | Genetic transformation method for DREB (Dehydration Responsive Element Binding) gene transformation Anthurium |
| CN104673716A (en) * | 2015-02-06 | 2015-06-03 | 海南大学 | Sfp gene-deleted biocontrol bacteria as well as fermentation technology and application thereof |
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| WO2005059112A1 (en) * | 2003-12-17 | 2005-06-30 | Kt & G Co., Ltd | The novel bacillus amyloliquefaciens ktgb0202 and control method of plant pathogenic funzi using that |
| CN102286527A (en) * | 2011-08-30 | 2011-12-21 | 南京农业大学 | Genetic transformation method for DREB (Dehydration Responsive Element Binding) gene transformation Anthurium |
| CN104673716A (en) * | 2015-02-06 | 2015-06-03 | 海南大学 | Sfp gene-deleted biocontrol bacteria as well as fermentation technology and application thereof |
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