CN108148794A - A kind of the bacillus subtilis DYr3.3 and preparation method and application of broad-spectrum antibacterial activity - Google Patents
A kind of the bacillus subtilis DYr3.3 and preparation method and application of broad-spectrum antibacterial activity Download PDFInfo
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Abstract
The invention discloses the bacillus subtilis DYr3.3 and preparation method and application of a kind of broad-spectrum antibacterial activity, antagonistic bacterium bacillus subtilis strain DYr3.3, CCTCC M 2018024, step is：（1）Root, is cut into the tissue of size, by 70% alcohol disinfecting, after aseptic water washing, is positioned over light culture in PDA culture medium by the purple branch rose root of acquisition, after culture, longer bacterial clump is transferred to scribing line in new PDA culture medium and isolates and purifies single bacterium colony；（2）After isolating and purifying, a kind of antagonistic bacterium bacillus subtilis strain DYr3.3, nucleotide sequence such as SEQ ID NO are obtained：Shown in 1, applications of the bacterial strain DYr3.3 in preparation treatment or prevention pear fruit rots medicine.Bacterial strain DYr3.3 can prevent the pear fruit caused by black rot of pear and anthracnose endanger from rotting by the DYr3.3 bacterium solutions that liquid fermentation obtains, to rotting with good protection effect for pear fruit, pear fruit is significantly inhibited to rot, anti-efficiency is widely used in the prevention of pear fruit rot disease up to 100%.
Description
Technical field
The invention belongs to technical field of plant disease biological control, are more particularly to a kind of withered grass bud of broad-spectrum antibacterial activity
Spore bacillus DYr3.3 also relates to a kind of preparation method of the bacillus subtilis DYr3.3 of broad-spectrum antibacterial activity, further relates to
A kind of purposes of the bacillus subtilis DYr3.3 of broad-spectrum antibacterial activity.
Background technology
Since the grown place of fruit and vegetable food is all far from city, and maturity period also Relatively centralized, need certain storage
And the haul-cycle time, it is lost after largely adopting during this period, particularly fruits and vegetables is rotten.It has been reported that there is 10%-30% in developed country
Fresh fruit be lost in after adopting and rot, in the developing country for lacking storing refrigerating equipment, rotten loss rate is up to 40%-
50%.At present to the prevention of this kind of disease still based on Agro-chemicals control, this not only generates people, animal and ecological environment
Seriously endanger, can also increase the residual quantity of toxic chemical substance in agricultural product, while also easily lead to plant to pathogen anti-medicine
The generation of property.In recent years, there is the chemical pesticide that researcher replaces parts of traditional using bacillus, be postharvest disease of fruits and vegetables
Prevention provides new research ideas and methods【Generation gentle model in field is green, controls the biology techniques of postharvest disease of fruits and vegetables, and botany is led to
Report, 2000,17 (3)：211-217】.
Often occur to rot during storage after pear fruit harvesting, adopt the rotten often by Target spot pathogen and charcoal of rear storage phase
Caused by subcutaneous ulcer germ is infected.At present, it is existing to study the apple decay that storage phase is controlled using bacillus, including using lichens bud
Spore bacillus prevents the canker of apple fruit as caused by Botryosphaeria berengeriana f. sp and anthrax bacteria【Ji Zhaolin etc., bacillus licheniformis is to apple
The inhibition of fruit Target spot pathogen and anthrax bacteria and its preventive and therapeutic effect to storage period ring rot of apple, Journal of Fruit Science, 2008,25
(2)：20 9~2 14】, handle Apple diseases prevention rate and reach 33.0%；Using bacillus amyloliquefaciens NCPSJ7 to adopting rear apple
Fruit ring spot is prevented【Yellow tinkling of pieces of jades etc., using bacillus amyloliquefaciens NCPSJ7 to adopting the biological control of rear ring rot of apple
Effect, Chinese food and nutrition 2015,21 (2)：20-24】；Rear ring rot of apple is adopted using bacillus subtilis B-903 controls,
10 times of liquid of inoculation B-903 bacterium solutions, 20 times of liquid treatment effects are preferable before germ intrusion, and the inhibition of 7d reaches 64.4-68.2%；
Germ intrusion morbidity after with B-903 handle inhibition be 13.5-25.8%【Zhao Baige etc., B-903 pairs of bacillus subtilis
The bacteriostasis and its control effect to disease of apple wheel line bacterium, Plant Pathology, 1996,27（3）213-214】.More than
Research mainly adopts rear ring rot of apple and anthracnose, but each bacterial strain effect is all not ideal enough using the control of various bacillus,
And working concentration is all relatively low（Stoste or 20 times slightly dilute）, it is impossible to meet the upper practical application needs of production.Also someone screening and
Rear black rot of pear is adopted using bacillus subtilis prevention, 21 bacillus subtilis are isolated to from pears sample, can be inhibited
Pear fruit is rotten after inoculation pears wheel line bacterium, and it is more than 40% to inhibit Spot expansion efficiency【Ding Fangbing screens and utilizes withered grass bud
Spore bacillus prevents black rot of pear, Jiangsu's agriculture journal, 2009,25 (5)：1002-1006】, it is also upper real far from production is met
Border application needs.
Invention content
The purpose of the invention is to provide a kind of broad-spectrum antibacterial activity bacillus subtilis (Bacillus subtilis) DYr3.3, which has broad-spectrum antifungal activity, to the main pathogen fungi of pear tree, including black rot of pear bacterium
（Botryosphaeria berengeriana）, pears anthrax-bacilus（IncludingColletotrichumfructicola、 Colletotrichumgloeosporioides）, pears rotten pathogenic bacteria（Valsamalivar. pyri）, pears stem blight bacterium
（Phomopsis fukushii）Deng）There is significant bacteriostatic activity.In addition, also to other common garden crop disease fungus,
Such as camellia colletotrichum（Colletotrichumcamelliae）, Phomopsis bacterium（Phomopsissp.）, Pestalotiopsis theae
（Pestalotiopsis_theae）, citrus Penicillium notatum（Penicilliumitalicum）It is respectively provided with good antagonistic activity.
Another object of the present invention is to be the provision of a kind of preparation side of the bacillus subtilis of broad-spectrum antibacterial activity
Method, it is easy to implement the method, it is easy to operate, after liquid fermentation, obtain convenient for the liquid preparation soluble in water using with storage endurance,
It is 4.41 × 10 to measure spore concentration7cfu/mL
A further object of the invention be the provision of a kind of bacillus subtilis of broad-spectrum antibacterial activity (Bacillus subtilis) applications of the DYr3.3 in preparation treatment or prevention pear fruit rots medicine, the fermented product pair of the bacterial strain
Pear fruit rots, with good protection effect, to significantly inhibit pear fruit and rot, preventive effect can reach 100%.
In order to realize above-mentioned purpose, the present invention uses following technical measures：
The study find that a bacillus subtilis strain DYr3.3, the bacterial strain can with strong inhibition pear fruit because of wheel line bacterium and
It rots caused by anthrax-bacilus, effect can reach 100%, have significant practical application potentiality.
A kind of preparation method of the bacillus subtilis of broad-spectrum antibacterial activity, step are：
（1）Purple branch rose root is acquired to rose in Hubei Province Huangshi Daye, root is cut into the tissue for being about 0.5 cm sizes, is passed through
Cross 70%（Volume ratio）1 min of alcohol disinfecting after aseptic water washing 2-3 times, is positioned over 24-26 DEG C of dark training in PDA culture medium
It supports, after cultivating 34-38h, longer bacterial clump is transferred to scribing line in new PDA culture medium and isolates and purifies single bacterium colony；
The preparation method of the PDA culture medium is：200 g potatos, 20 g sucrose, 15 g agar powders are dissolved in 1000 mL
Distilled water, tune pH value to 6.8-7.2 or so；
（2）After isolating and purifying, obtain it is a kind of from purple branch rose root antagonistic bacterium bacillus subtilis (Bacillus subtilis) bacterial strain DYr3.3, nucleotide sequence such as SEQ ID NO：Shown in 1, bacterial strain DYr3.3 was on January 12nd, 2018
China typical culture collection center (CCTCC) is preserved in, deposit number is CCTCC M 2018024, and DYr3.3 is in tablet pair
It stands erect in testing to pears wheel line bacterium（See Fig. 1）And various pathogens（See Fig. 3）With very strong antagonistic ability, rot to have to pear fruit
There is good preventive effect；
A kind of bacillus subtilis DYr3.3 of broad-spectrum antibacterial activity is rotted in preparation treatment or prevention pear fruit in medicine
Using step is：
A, picking DYr3.3 bacterial strain single bacterium colonies under gnotobasis are inoculated in equipped with equipped with the most suitable culture solution A of 90mL（3.0%（Matter
Measure volume ratio）Glucose, 0.5%（Mass volume ratio）Yeast extract powder, 0.3%（Mass volume ratio）Sodium chloride, 0.1%（Matter
Measure volume ratio）Magnesium sulfate adjusts pH values 6.0~6.5）250mL triangular flasks in, 28 °C, 24 h of 150r/min shaken cultivations,
Obtain seed culture bacterium solution.
B, prepare 500ml triangular flasks 10, per bottled 200 mL culture solution A, every bottle according to 1:100 ratio adds in step
The seed culture bacterium solution that A is obtained is placed in large amplitude cryostat shaking table, 150 r/min shaken cultivations, 48 h under 28 °C,
Acquisition DYr3.3 zymotic fluids, a concentration of 4.89 × 106cfu/mL。
C, the DYr3.3 bacterial strain fermentation liquors that step B is obtained are taken, 100 times is diluted with clear water, is dragged for after pear fruit is impregnated 30 s
Go out, dry, low temperature（4°C）Or room temperature（It is 20-25 °C, same as below）Storage.
Bacterial strain DYr3.3 can be prevented by the DYr3.3 bacterium solutions that liquid fermentation obtains because of black rot of pear and anthracnose harm
Caused by pear fruit rot, can be applied to the prevention of pear fruit rot disease.
Compared with prior art, the present invention haing the following advantages and effect：
（1）Often occur to rot during storage after pear fruit harvesting, adopt the rotten often by Target spot pathogen and charcoal of rear storage phase
Caused by subcutaneous ulcer germ is infected.It there is no the bacillus category biological prevention and control agent that effectively prevention pear fruit rots, bacillus subtilis at present
DYr3.3, which has, is applied to the remarkable result that prevention pear fruit rots, and has the advantages that be applied to preserving fruit and vegetable utilizing and effect.
（2）The bacillus subtilis strain of commercialized development, which is mainly used for field crop field diseases, to be prevented, and non-fruit
Postharvest diseases are prevented, and the bacillus subtilis B-916 bacterial strains as Jiangsu Academy of Agricultural Sciences's Plant Protection Institute is found have carried out
Agriculture chemical registration, applied to rice sheath blight disease field control；Agricultural University Of Nanjing biocontrol microorganisms B3 (trade name wheat Fengning) is applied to small
Wheat banded sclerotial blight field control.Therefore, bacterial strain DYr3.3 has answering different from other commercialization bacillus subtilis strains
Use direction.
（3）Early period, someone was screened applied to the bacillus subtilis for preventing Target spot pathogen.For example, there is researcher
21 bacillus subtilis strains are obtained from the separation of pears sample, Spot expansion can be inhibited, efficiency is about 40%【It refers to：Fourth
Fragrant soldier screens and prevents black rot of pear, Jiangsu's agriculture journal, 2009,25 (5) using bacillus subtilis：1002-1006】；
Biocontrol microorganisms 11 are 23.83% to the preventive effect of black rot of pear【It refers to：Liu Youzhou etc., sodium bicarbonate（NaHCO3）Biocontrol microorganisms are prevented
Adopt the influence of rear black rot of pear, Journal of Fruit Science, 2010,27 (5): 757 – 763】；2 bacillus subtilis Sf-19 and
Sf-28 stostes are 71.97% and 64.54% to the indoor inhibition efficiency of black rot of pear bacterium【It refers to：Zhang Lili etc., 2 plants of withered grass buds
Indoor inhibiting effect research of the spore bacillus to black rot of pear bacterium, Journal of Fruit Science, 2010,27（5）：823-827】.Also there is similar grind
Study carefully and rear ring rot of apple is adopted, but result shows that the bacterial strain control effect is bad using bacillus subtilis control, only at 10 times
The inhibition of 7d reaches 64.4-68.2 under liquid, 20 times of liquid processing【Zhao Baige etc., bacillus subtilis B-903 is to apple wheel
The bacteriostasis of line bacterium and its control effect to disease, Plant Pathology, 1996,27（3）213-214】.In conclusion
Effect is not obvious the bacillus subtilis of separation early period in the prevention experiment of black rot of pear after prevention is adopted, therefore, should
DYr3.3 bacterial strains have preferably prevention pear fruit decomposition than the bacillus subtilis strain obtained early period.
（4）Also there is the apple decay that research controls storage phase using other bacillus, including using lichens gemma bar
Bacterium and bacillus amyloliquefaciens the prevention canker of apple fruit as caused by Botryosphaeria berengeriana f. sp and anthrax bacteria, but obtained bacterial strain is prevented
Sick effect is not notable【See：Ji Zhaolin etc., bacillus licheniformis is to the inhibition of Botryosphaeria berengeriana f. sp and anthrax bacteria and its to storage
The preventive and therapeutic effect of Tibetan phase ring rot of apple, Journal of Fruit Science, 2008,25 (2)：20 9~2 14；Yellow tinkling of pieces of jades etc., using solution starch
Bacillus NCPSJ7 acts on the biological control for adopting rear ring rot of apple, Chinese food and nutrition 2015,21 (2)：20-24】.
Therefore, bacillus subtilis strain DYr3.3 bacterial strains have preferably prevention than the other Bacillus species bacterial strains obtained early period
Pear fruit decomposition.
Description of the drawings
The form and the function and effect of inhibition pears wheel line bacterium that Fig. 1 is a kind of bacillus subtilis strain DYr3.3.
Wherein, it is pears wheel line bacterium among PDA culture medium（Botryosphaeria berengeriana）Bacterial strain, display life
Length is inhibited by surrounding bacterial strain；It is multiple bacterial strain DYr3.3 bacterium colonies around PDA culture medium.
Fig. 2 is a kind of linear between bacillus subtilis strain DYr3.3 differences light absorption value and concentration under 600nm wavelength
Relational graph.
The figure is that picking bacterial strain DYr3.3 single bacterium colonies are put into culture medium A（3.0% glucose, 0.5% yeast extract powder,
0.3% sodium chloride, 0.1% magnesium sulfate adjust pH values 6.0~6.5）In, after 28 DEG C of 48 h of culture, according to 1,2,3,4,5,10,
20th, it is diluted for 30,40,50,100 times, measures the light absorption value under 600 nm wavelength.50 μ L is taken to be diluted to 10−5 、10−6
Times, it is seeded on most suitable solid medium and carries out coated plate, 28 °C of constant incubator is positioned over after sealing and is cultivated, it is fixed
Phase observation is measured again until there is clear denumerable bacterium colony.OD600 light absorption values and corresponding Fungal biodiversity are measured, display is inhaled
Light value（x）Be proportionate with biomass (y), linear equation be y=45,607,035x-831,951（R2=0.9884）.
Fig. 3 is a kind of bacillus subtilis bacterial strain DYr3.3 in PDA culture medium to the antibacterial effect of the face-off of 12 fungal bacterial strains
Fruit is schemed.
Tablet center is the DYr3.3 bacterium solutions of scribing line, and both sides are the hypha,hyphae block of inoculation.CK-To be not added with bacterial strain
DYr3.3 only adds the control of hypha,hyphae block.Fungi and bacterial strain are labeled in above tablet.
Fig. 4 is that a kind of Paenibacillus polymyxa bacterial strain DYr3.3 in PDA culture medium presses down the face-off of 12 fungal bacterial strains
Bacterium width block diagram.Ordinate is antibacterial width, and abscissa is measures fungi and bacterial strain.Significant difference analysis is using SPSS
Statistics 21.0 (WinWrap Basic; http://www.winwrap.com) carry out significance difference analysis
（Duncan ' s test, P=0.05）.
Fig. 5 inhibits pear fruit to rot to represent design sketch for a kind of DYr3.3.
A, left figure " CK+ " are inoculation pears wheel line bacterium mycelia block（On）With pears anthrax-bacilus mycelia block（Under）Fruit morbidity picture；
After right figure " DYr3.3 " is impregnates DYr3.3 zymotic fluids, it is inoculated with pears wheel line bacterium mycelia block（On）With pears anthrax-bacilus mycelia block（Under）Pears
The picture that rots does not occur for fruit rot；B is individually inoculated with pears wheel line bacterium mycelia block and pears anthrax-bacilus mycelia block and impregnates DYr3.3
Scab length block diagram after both bacterium is inoculated with after zymotic fluid.
Specific embodiment
Embodiment 1：
A kind of preparation method of the bacillus subtilis of broad-spectrum antibacterial activity, step are：
（1）Purple branch rose root is acquired to rose in Hubei Province Huangshi Daye, root is cut into the tissue for being about 0.5 cm sizes, is passed through
Cross 70%（Volume ratio）1 min of alcohol disinfecting after aseptic water washing 2-3 times, is positioned over 25 DEG C of light cultures in PDA culture medium, training
After supporting 36h, longer bacterial clump is transferred to scribing line in new PDA culture medium and isolates and purifies single bacterium colony；
The preparation method of the PDA culture medium is：200 g potatos, 20 g sucrose, 15 g agar powders are dissolved in 1000 mL
Distilled water adjusts pH value to 7.0 or so；
（2）After isolating and purifying, obtain one plant from purple branch rose root antagonistic bacterium bacillus subtilis (Bacillus subtilis) bacterial strain DYr3.3, nucleotide sequence such as SEQ ID NO：Shown in 1, bacterial strain DYr3.3 was on January 12nd, 2018
China typical culture collection center (CCTCC) is preserved in, deposit number is CCTCC M 2018024, and DYr3.3 is in tablet pair
It stands erect in testing to pears wheel line bacterium（See Fig. 1）And various pathogens（See Fig. 3）With very strong antagonistic ability, rot to have to pear fruit
There is good preventive effect；
The Molecular Identification of Antagonistic Fungi DYr3.3:
Extract the genomic DNA of antagonistic bacterium DYr3.3【It refers to：Wang Ruixia etc., the identification of bacterial ring rot o potato biocontrol bacterial strain P1,
Protection effect and growth-promoting functions research, Plant Pathology, 2010,40（1）：66 - 73】, using the DNA of extraction as masterplate, adopt
Use BSF（5′-AGAGTTTGATCCTGGCTCAG-3′）And BSR（5′-AAGGAGGTGATCCAGCCGCA-3′）Primer pair 16S
RDNA is expanded, it is contemplated that size 1500bp.
PCR reaction systems：10×PCR buffer（Containing Mg2+）2.5 μ L, 10mmol/L dNTPs 0.5 μ L, 10 μ
Mol is homologous and complementary primer each 0.5 μ L, Taq DNA polymerases, 0.2 μ L, DNA template, 1 μ L, 19.8 μ L of deionized water.
PCR reaction conditions are：Then 95 DEG C of pre-degeneration 4min are denaturalized 30s, 55 DEG C of annealing 30s, 72 DEG C of extensions for 94 DEG C
90s, 35 cycles, 72 DEG C re-extend 5min, 16 DEG C of terminations.
PCR product is all analyzed through 1% agarose gel electrophoresis, and target fragment is served to the raw work bioengineering skill in sea after purification
Art Services Co., Ltd is sequenced.Sequence in the sequence of acquisition and NCBI databases is subjected to similarity analysis using BLASTn,
The result shows that：The 16S rDNA sequences of bacterial strain DYr3.3 and bacillus subtilis（Bacillus subtilis）The 16S of OPP3 1
RDNA sequences（The sequence number of logging in：JQ308571.1）Homology is 99%（coverage 98%, E value 0.0,
identity 99%）.
Microbial identification：
State Key Laboratory of Agricultural Microbiology is sent to carry out Biolog microbial identifications bacterial strain, carry out 71
Kind utilization of carbon source rate and 23 kinds of chemosensitivities measure.Key step is as follows：Bacterial strain DYr3.3 is subjected to 4th area on BUG tablets
Scribing line purifying culture after 33 DEG C of dark are incubated overnight, are chosen single bacterium and is fallen in inoculation liquid IF-B and mixing, be then seeded into Biolog
In 96 hole identification plates, 100 μ L are inoculated with per hole, sealing identification plate is placed in 33 DEG C and carries out light culture 16-24 hours, read later
Number.As a result show bacterial strain DYr3.3 withBacillus atrophaeus/subtilisThat is atrophy bacillus and bacillus subtilis
Bacterium similitude highest (PROB 0.575；SIM 0.575； DIST 6.258； Organism Type GP-Rod-SB；
Species Bacillus atrophaeus/subtilis).Comprehensive Molecular Identification and Biolog qualification results, DYr3.3 is true
It is set to bacillus subtilis（Bacillus subtilis）.
The bacterium colony and morphological feature of Antagonistic Fungi DYr3.3
On LB tablets, bacillus subtilis (Bacillus subtilis) DYr3.3 bacterial strains bacterium colony it is irregular, edge is normal
In irregular wavy protrusion, flat, milky is opaque（See Fig. 1）.
The culture of Antagonistic Fungi DYr3.3：
The condition of culture of DYr3.3 bacterial strains is：Bacterial strain activation is using PDA culture medium, i.e. 200 g potatos, 20 g sucrose, 15 g
Agar powder is dissolved in 1000 mL distilled water, pH value 7.0 or so.Appropriate bacterium solution is taken to carry out scribing line culture in point PDA culture medium.It connects
28 °C of constant incubator light cultures are positioned over after kind, until there is bacterium colony.
It is measured using culture medium A for the fermented and cultured and spore count purpose of bacterial strain DYr3.3, the specific formula of culture medium A
For：3.0% glucose（Mass volume ratio）, 0.5% yeast extract powder（Mass volume ratio）, 0.3% sodium chloride（Quality volume
Than）, 0.1% magnesium sulfate（Mass volume ratio）, adjust pH to 6.0~6.5；Wherein solid medium adds 7~8g of agar（Often
500mL culture mediums）, culture medium is in 121 DEG C of 20 min of high pressure steam sterilization.
The zymotic fluid obtained will be cultivated, culture medium A is added to be diluted to different multiples, obtain extension rate for 1,2,3,4,5,
10th, 20,30,40,50,100 times of sample bacteria suspension, and using culture medium A as blank control.Use UITROSPEC nucleic acid eggs
White detector measures different extension rate sample OD under 600 nm of wavelength600 Value.In order to improve the accuracy of experimental data,
Prepare 3 groups of Duplicate Samples.
Another prepare is diluted to 10−3 、10−4 、10−5 、10−6 Sample bacteria suspension again, it is fixed for spore count range estimation.Take 50
μ L are diluted to 10−5 、10−6 Times DYr3.3 samples bacteria suspension and be diluted to 10−4 、10−5 Bacteria suspension again is seeded in respectively
Coated plate is carried out on most suitable solid medium, 28 °C of constant incubator is positioned over after sealing and is cultivated, routine observation until
There is clear denumerable bacterium colony to be measured again.Accuracy is tested to improve, each extension rate sample does 5 repetitions.It measures
OD600 light absorption values and Fungal biodiversity linear equation are y=45,607,035x-831,951（R2=0.9884）（Fig. 2）.
The antagonistic experiment of Antagonistic Fungi
Antagonistic activities of the evaluation bacterial strain DYr3.3 to fungi is sieved using tablet face-off method：In PDA culture medium, 10 μ L is taken to shake training
Bacterium solution scrawle in the middle of culture dish, connect the mycelia block of a diameter of 5 mm at 4 cm from bacterium line, unrestraint be set
Bacterium processing is control, and each processing repeats 3 culture dishes, in 25 DEG C of light cultures, when control group just covers with whole culture dish, is measured each
Inhibition zone size is handled, its antagonistic effect is judged according to the size for whetheing there is antibacterial band and antibacterial band.
It tests antagonistic strain and includes black rot of pear bacterium（Botryosphaeria berengeriana）, fruit life anthrax-bacilus
（Colletotrichumfructicola）, pears rotten pathogenic bacteria（Valsamali var. pyri）, pears stem blight bacterium
（Phomopsis fukushii）, camellia colletotrichum（Colletotrichumcamelliae）, Pestalotiopsis theae
（Pestalotiopsis_theae）, Phomopsis bacterium（Phomopsissp.）, Mucor（Mucorracemosus）It is Italian green
Mould（Penicilliumitalicum）And bread mold（Rhizopusnigricans）, these bacterium are by this laboratory early period
It is separating obtained.The results show that DYr3.3 in addition to saprophytic bacteria Mucor and bread mold without apparent bacteriostasis other than, to it is other cause a disease it is true
Bacterium has strong bacteriostasis, and scope of restraining fungi is 5.3-11.5 mm, and has conspicuousness to the fungistatic effect of different pathogenic bacteria
Difference.
Embodiment 2
A kind of bacillus subtilis of broad-spectrum antibacterial activity (Bacillus subtilis) preparing or preventing pear fruit rot disease
Application in drug, step are：
A, picking DYr3.3 bacterial strain single bacterium colonies under gnotobasis are inoculated in equipped with equipped with the most suitable culture solution A of 90mL（3.0%（Matter
Measure volume ratio）Glucose, 0.5%（Mass volume ratio）Yeast extract powder, 0.3%（Mass volume ratio）Sodium chloride, 0.1%（Matter
Measure volume ratio）Magnesium sulfate adjusts pH values 6.0~6.5）250mL triangular flasks in, 28 °C, 24 h of 150r/min shaken cultivations,
Obtain seed culture bacterium solution.
B, prepare 500mL triangular flasks 10, per bottled 200 mL zymotic fluids, every bottle according to 1:100（Volume ratio）Ratio
Seed culture bacterium solution is added in, is placed in large amplitude cryostat shaking table, 150 r/min shaken cultivations, 48 h, is obtained under 28 °C
DYr3.3 zymotic fluids, a concentration of 4.89 × 106cfu/mL。
Take bacillus subtilis (Bacillus subtilis) DYr3.3 bacterial strain fermentation liquors, 100 times are diluted with clear water, it will
Pear fruit is pulled out after impregnating 30 s, is dried, low temperature（4-8℃）Or room temperature（20-25 DEG C）Storage.
Embodiment 3：
The prevention pear fruit of Antagonistic Fungi rots to test
Bacillus subtilis strain DYr3.3 is diluted into 100 times of liquid, obtains a concentration of 4.89 × 106Bacteria suspension, then will be from super
Buy full ripe pear fruit in city（Hebei imperial crown pears,Pyrus bretschneideriRehd. var. Huangguanli）
About 30 s in prepared bacteria suspension is separately immersed in, its surface is made sufficiently uniformly to be stained with bacterium, takes out dry later.Each
6 repetitions of processing group, in the black rot of pear bacterium bacteria cake of the equidistant inoculation activation of fruit waist（5 mm of diameter）, then sterile water will be used
The cotton piece for the moistening impregnated is covered at inoculation.Separately 2 pears impregnated without bacteria suspension are taken as CK+Control group.Place
The pears managed are respectively placed in the plastic pallet for being covered with one layer of moistening gauze and preservative film, keep having in disk at any time higher opposite
Humidity finally with preservative film sealed plastic disk, is placed under 30 DEG C of environment and cultivates.Result is recorded after positive control morbidity is serious.
The results show that it is inoculated with pears wheel line bacterium after spraying fruit using DYr3.3 zymotic fluids（Botryosphaeria berengeriana）Mycelia block carries out diseases prevention experiment；Inoculation mycelia block is as control after spraying sterile water simultaneously.The results show that
After inoculation 2 days, the control group that mycelia block is inoculated with after spray clear water starts the bacterium that falls ill, and scab then gradually extends, until 5 days, hair
Disease fruit is substantially all in fact to rot, and DYr3.3 fermentation liquor treatments fruit is without morbidity, and result is similar in triplicate.
Meanwhile it is inoculated with colletotrichum gloeosporioides Penz after fruit will be sprayed using DYr3.3 zymotic fluids
（Colletotrichumgloeosporioides）Pears wheel line bacterium（Botryosphaeria berengeriana）Mycelia block into
Row diseases prevention is tested, while sprays sterile water inoculation mycelia block as control, and 6 repetitions are united after control group fruit rots completely
Count result.The results show that being inoculated with after being inoculated with 7 days, after spray clear water, the morbidity of mycelia block control group is serious, and morbidity fruit is substantially all
It rots, and bacterial strain DYr3.3 handles fruit without morbidity（See Fig. 5）.
Sequence table
<110>Hua Zhong Agriculture University
<120>A kind of the bacillus subtilis DYr3.3 and preparation method and application of broad-spectrum antibacterial activity
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1488
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gccaaggtct gggagtctat aatgcagtcg agcggacaga tgggagcttg ctccctgata 60
ttagcggcgg acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg 120
ggaaaccggg gctaataccg gatggttgtc tgaaccgcat ggttcagaca taaaaggtgg 180
cttcggctac cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct 240
caccaaggcg acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300
acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360
acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga 420
agaacaagtg ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg 540
gcgtaaaggg ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg 600
gagggtcatt ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta 660
gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt 720
aactgacgct gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca 780
cgccgtaaac gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac 840
gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900
gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960
ggtcttgaca tcctctgaca atcctagaga taggacgtcc ccttcggggg cagagtgaca 1020
ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080
cgcaaccctt gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga 1140
caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200
cacgtgctac aatggacaga acaaagggca gcgaaaccgc gaggttaagc caatcccaca 1260
aatctgttct cagttcggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag 1320
taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380
acaccacgag agtttgtaac acccgaagtc ggtgaggtaa cctttatggt agccagccgc 1440
cgaaggtggg acagatgatt ggggtgaagt cgaagcaacg ttgcatag 1488
Claims (3)
1. a kind of bacillus subtilis DYr3.3 of broad-spectrum antibacterial activity, it is characterised in that：Antagonistic bacterium bacillus subtilis
(Bacillus subtilis) bacterial strain DYr3.3, deposit number is CCTCC M 2018024.
2. a kind of bacillus subtilis DYr3.3 of broad-spectrum antibacterial activity according to claim 1, it is characterised in that：It is described
Antagonistic bacterium bacillus subtilis DYr3.3 bacterial strains in contain SEQ ID NO：Nucleotide sequence shown in 1.
3. a kind of bacillus subtilis DYr3.3 of broad-spectrum antibacterial activity described in claim 1 is preparing treatment or prevention the operatic circle
Application in real rotten medicine, step are：
A, picking DYr3.3 bacterial strain single bacterium colonies under gnotobasis are inoculated in the 250mL triangular flasks equipped with the most suitable culture solution A of 90mL
In, 28 °C, 24 h of 150r/min shaken cultivations obtain seed culture bacterium solution；
B, prepare 500ml triangular flasks 10, per bottled 200 mL culture solutions A, every bottle is 1 according to volume ratio:100 ratio adds in
The seed culture bacterium solution that step A is obtained is placed in large amplitude cryostat shaking table, 150 r/min shaken cultivations under 28 °C
48 h, acquisition DYr3.3 zymotic fluids, a concentration of 4.89 × 106cfu/mL；
C, the DYr3.3 bacterial strain fermentation liquors that step B is obtained are taken, 100 times is diluted with clear water, pulls out, dry in the air after pear fruit is impregnated 30 s
It is dry, low temperature or room storage；
The configuration method of the culture solution A is：Mass volume ratio is 3.0% glucose, and mass volume ratio is 0.5% ferment
Female extract powder, mass volume ratio are 0.3% sodium chloride, and mass volume ratio is 0.1% magnesium sulfate, adjust pH values 6.0~
6.5。
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| CN114410528A (en) * | 2022-01-25 | 2022-04-29 | 山西农业大学食品科学与工程学院(山西省农业科学院农产品贮藏保鲜研究所) | Biocontrol bacterial liquid for Yulu bergamot pears during storage period and preparation method and application thereof |
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