CN107164278A - One plant of endophytic Bacillus subtilis YC01 and its application in preventing and treating Oil Tea Anthracnose - Google Patents
One plant of endophytic Bacillus subtilis YC01 and its application in preventing and treating Oil Tea Anthracnose Download PDFInfo
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Abstract
Application the present invention relates to one plant of endophytic Bacillus subtilis and its in preventing and treating Oil Tea Anthracnose.Endophytic Bacillus subtilis (Bacillus subtilis) YC01 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and deposit number is：CGMCC No.14186.Endophytic Bacillus subtilis and its albumen and the lipopeptid extract of the present invention can effectively suppress the growth of Oil Tea Anthracnose bacterium, can be applied to the biological control of Oil Tea Anthracnose in oil tea production.
Description
Technical field
The invention belongs to the biological prosthetic field of plant disease, specifically, it is related to one plant of endophytic Bacillus subtilis YC01
And its albumen and lipopeptid extract are in the application in preventing and treating Oil Tea Anthracnose.
Background technology
Oil tea is the distinctive woody oleiferous plants crop of China, south China area is distributed mainly on, with oil palm, olive and oil
The big woody edible oil seeds in coconut and the referred to as world four, in the world about 90% camellia seed oil yield come from China.Oil tea produces
Industry is the specialty industries of Hunan Province's agricultural and forestry production, and Hunan Province is the oil tea place of production and maximum tea oil consumption area of largest domestic.Root
According to《Hunan Province's camellia oleiferaindustry development plan (2011-2020)》, to the year two thousand twenty, Hunan will build up 22,000,000 mu of high yield oil teas
Garden, more than 40,000,000,000 yuan of annual value of production, the supporting industry as Hu-nan's agriculture industry.Oil Tea Anthracnose is the most important disease of oil tea
One of evil, its pathogen is mainly colletotrichum gloeosporioides Penz (Colletotrichum gloeosporioides), is distributed widely in me
South each province of state, can cause the serious bud drop of tea oil tree and shedding, fruit drop rate is up to 50% generally 20% or so when serious.
The current preventing and treating to Oil Tea Anthracnose is mainly controlled by planting resistant variety and chemical prevention, but long-term change
Learning preventing and treating causes Pest-resistant problem increasingly serious, and chemical pesticide residual causes serious harm, such as causes residual hazard, dirt
Environment is contaminated, very big threat is caused to health, ecological environment, is unfavorable for the sustainable development of camellia oleiferaindustry.Due to life
Thing prevents and treats the favorable compatibility with environment, and new thinking and selection are provided to plant pest management.At present using more
Biocontrol bacteria mainly includes bacillus (Bacillus), pseudomonad (Pseudomonas), soil radiation bacillus
(Agrobacterium radiobacter) etc..Bacillus subtilis (Bacillus subtilis) is a kind of mesophilous good
The sporiferous Gram-positive bacillus of oxygen, is the most representational strain of Bacillaceae bacillus, this interior life
The bacillus of gemma is also the type strain for studying gram positive bacterial cell biology.Bacillus subtilis can conduct
The good source of crop disease biological control, is with a wide range of applications.
The content of the invention
The invention provides one plant of endophytic Bacillus subtilis bacterial strain, the good of upper Oil Tea Anthracnose can be produced as oil tea
Good biological control microbial inoculum, and provide foundation for the field biological control of Oil Tea Anthracnose.
The isolated interior raw biocontrol microorganisms of leaf are good for from oil tea the invention provides one plant, bacillus subtilis is identified as
(Bacillus subtilis), numbering YC01 was preserved in Chinese microorganism strain preservation conservator on May 24th, 2017
Can common micro-organisms center (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism
Research institute, postcode 100101), preserving number is CGMCC No.14186.
Bacterial strain of the present invention has preferable antagonistic effect to Oil Tea Anthracnose bacterium, is given birth to according to strain morphology feature, physiology
Change feature, 16S rDNA sequence homology analysis, bacterial strain YC01 is accredited as bacillus subtilis (Bacillus
subtilis)。
Bacillus subtilis YC01 of the present invention microbial inoculum can use LB culture mediums, be cultivated according to a conventional method.
The present invention also provides the microbial inoculum containing the bacillus subtilis YC01.
The preparation method of the microbial inoculum is：LB culture medium inoculated strains YC01,160-200r/min, under the conditions of 25-28 DEG C
Fermented and cultured 4-8 days.Liquid preparation or solid pharmaceutical preparation are prepared into according to different application aspects.
Specifically, a kind of preparation method of the liquid bacterial agent containing the bacillus subtilis YC01 includes：With 250mL tri-
The bottled 100mL LB liquid mediums in angle, conventional sterilant, after cooling on superclean bench, picking YC01 thalline are inoculated with into fermentation
In triangular flask, shaken cultivation, cultivates 24h and is transferred to ferment tank, 28 DEG C, 160r/min at once by 28 DEG C under the conditions of 160r/min
Under the conditions of cultivate 96h stuck fermentations.Zymotic fluid is dispensed using plastic bottle or polybag.
Present invention additionally comprises above-mentioned endophytic Bacillus subtilis (Bacillus subtilis) YC01, its metabolite, egg
White crude extract, lipopeptid crude extract and the application containing its microbial inoculum in preventing and treating Oil Tea Anthracnose.
Endophytic Bacillus subtilis (Bacillus subtilis) YC01 of the present invention metabolite, albumen is slightly carried
Thing, lipopeptid crude extract can be prepared by this area conventional method.
Bacterial strain of the present invention can be applied to produce the biological control of Oil Tea Anthracnose, and cooperate with preventing and treating oily with chemical pesticide
Tea anthracnose；Prevention effect is notable.
Brief description of the drawings
Fig. 1 is bacterial strain YC01 of the present invention colonial morphology photo；
Fig. 2 represents flat board antagonistic effects of the bacterial strain YC01 of the present invention to Oil Tea Anthracnose bacterium；
Fig. 3 is the phylogenetic tree of bacterial strain YC01 bacterial strains of the present invention；
Fig. 4 represents prevention effect of albumen (DB) extract to Oil Tea Anthracnose in bacterial strain YC01 zymotic fluids of the present invention；
Fig. 5 represents prevention effect of lipopeptid (ZT) extract to Oil Tea Anthracnose in bacterial strain YC01 zymotic fluids of the present invention.
Bacterial strain YC01 of the present invention 16S rDNA sequences are shown in SEQ NO.3.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention
In the case of essence, the modifications or substitutions made to the inventive method, step or condition belong to the scope of the present invention.
Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.
The separation and identification of the antagonistic strain of embodiment 1
Be good for isolated one plant of leaf from oil tea using plate streak has in preferable antagonism to Oil Tea Anthracnose bacterium
Raw biocontrol microorganisms YC01, YC01 colonial morphology is as shown in Figure 1.
1st, culture medium：
LB culture mediums：Dusty yeast 5g, peptone 10g, sodium chloride 10g (solid medium adds agar 15g), 1000mL go from
Sub- water, pH7.0,121 DEG C of high pressure steam sterilization, 30min.
2nd, interior raw Antagonistic Fungi is isolated and purified
Choose oil tea and be good for leaf, rinse blade surface well with water, choose appropriate strong leaf texture, 4mm is cut into after weighing2's
Tissue block, soaks 60s in 75% ethanol, then soaks 40s in 0.1% mercuric chloride solution, places into the immersion of 75% ethanol
30s, takes out strong leaf texture's block aseptic water washing 4 times, thoroughly removes thimerosal.In sterile mortar add 10mL sterilized waters and
The quartzite sand grind sterilized a little stands 30min, homogenate is carried out into 10 times of serial dilutions with sterilized water into homogenate, capping,
0.1mL dilutions are drawn respectively with liquid-transfering gun to the LB flat boards sterilized, root after incubator culture, 36-48h is put into after smearing uniformly
According to colonial morphology, color bacterium colony different with size picking, and purification storage is carried out on LB flat boards respectively.
Flat board face-off method primary dcreening operation：A diameter of 6mm Oil Tea Anthracnose bacterium is put in PDA plate center, then with connecing collarium
Suspension equidistant (the anomaly plate center 3cm) point for dipping tested bacteria is connect in 2-3 kind endogenetic bacterias, incubator under the conditions of 28 DEG C
The presence or absence of antibacterial band of observed and recorded and size, are repeated 3 times after culture, 4d, choose the larger bacterium colony of antibacterial bandwidth and carry out secondary screening.
Fermentation method secondary screening：After the interior raw Antagonistic Fungi of primary dcreening operation is activated on LB flat boards, it is put into connecing collarium and pipetting a ring
In 50mL LB liquid mediums, 28 DEG C, shaken cultivation 72h under the conditions of 160rpm.Inoculum is used after removing thalline through centrifugation
0.22 μm of biofilter filtering, 1 is pressed by filtrate and about 50 DEG C of PDA culture medium:19 ratios pour into culture dish after mixing, and cool down
A diameter of 6mm Oil Tea Anthracnose bacterium bacteria cake (or rice sheath blight disease) is put into flat board center afterwards, blank pair is used as with sterilized water
According to, after 4d with crossing method measure germ colony diameter, select the best bacterial strain of fungistatic effect, save backup.
Growth of pathogenic bacteria inhibiting rate (%)=(control colony diameter-processing colony diameter)/control colony diameter * 100%
Fermentation method secondary screening result shows that YC01 zymotic fluids are to the inhibition of Oil Tea Anthracnose bacterium up to more than 85% (figure
2)。
3rd, bacterial strain YC01 identification
YC01 morphological features：YC01 bacterial strains bacterium colony is coarse opaque in LB culture mediums upper surface, dirty white or slightly yellow (figure
1).ESEM hypothallus is in rod-short.
YC01 bacterial strain physio-biochemical characteristics：Gram-positive, catalase, oxidizing ferment, VP experiments, glucose utilization,
Mannitol hydrolysis, Starch Hydrolysis, gelatin liquefaction test are positive.
Molecular Identification：Degradation bacteria DNA is extracted using kit, amplimer used is synthesized by Shanghai life work：Sense primer
For 5 '-CAGAGTTTGATCCTGGCT-3 ', anti-sense primer is 5 '-AGGAGGTGATCCAGCCGCA-3 '.
Amplification reaction system is：10×Buffer(Mg2+) 2.5 μ L, dNTPs 1 μ L, each 0.5 μ L of primer, thallus DNA 0.5
The μ L of μ L, Taq archaeal dna polymerase 0.2, plus deionized water is to 25 μ L.
PCR reaction conditions：94 DEG C of 4min, 94 DEG C of 45s, 55 DEG C of 40s, 72 DEG C of 60s；30 circulations；Most after 72 DEG C of reparations
Extend terminating reaction under the conditions of 10min, 4 DEG C.
PCR primer using Ago-Gel DNA QIAquick Gel Extraction Kits carry out purpose fragment recovery, recovery product examining order by
Shanghai Sheng Gong companies complete.Sequencing result is compared into GenBank databases.By the 16S rDNA sequences of YC01 bacterial strains
(i.e. SEQ NO.3) common 1509bp logs on GenBank and is compared and phylogenetic tree construction (Fig. 3), is accredited as withered grass
Bacillus (Bacillus subtilis).The bacterium is preserved in Chinese microorganism strain preservation management on May 24th, 2017
Committee's common micro-organisms center (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences is micro-
Biological study institute, postcode 100101), preserving number is CGMCC No.14186.
Prevention effect of the embodiment 2YC01 bacterial strains to Oil Tea Anthracnose
The YC01 bacterial strains that embodiment 1 is separated are transferred on fresh LB flat boards and activated, and subsequent picking colony is transferred to liquid
Body LB culture medium shaken cultivation 4d, carry out centrifugation by zymotic fluid and remove thalline, supernatant is standby.
The consistent healthy oil tea Newborn Leaves piece of leaf age is gathered, sterilized water is used after carrying out surface sterilization to blade with 75% alcohol
Blade surface residual alcohol rinse is clean, it is placed in culture dish, oil tea blade petiole is wrapped up with absorbent cotton, and is protected with sterilized water
It is wet.With the upper surface of the inoculation pricking wound blade of sterilizing but do not pierce through, each blade is stabbed at one.Oil Tea Anthracnose bacterium is transferred to
Fresh PDA plate is activated, and continuous culture 4-5d, beats in colony edge card punch and take diameter 6mm's under the conditions of 28 DEG C
Mycelia block, place is stabbed by inoculated by hypha block in oil tea blade, each 10 blades of processing.Setting CK groups altogether, (oil tea blade is stabbed
And be inoculated with anthrax bacteria mycelia block), Control groups (healthy Camellia Leaves piece and be not inoculated with anthrax bacteria mycelia block), treatment group (oil
After tealeaves piece is stabbed, blade is uniformly smeared with YC01 fermented liquid supernatant liquid, then inoculation anthrax bacteria mycelia block is in stabbing place).
Oil tea blade incidence is observed by day and record statistics after inoculation 5d.It the results are shown in Table 1.
Prevention effect of the bacterial strain YC01 zymotic fluids of table 1 to Oil Tea Anthracnose
| Handle the number of sheets | The morbidity number of sheets | The incidence of disease | Prevention effect | |
| CK | 10 | 9 | 90% | -- |
| Control | 10 | 0 | 0 | -- |
| YC01 processing | 10 | 2 | 20% | 77.8% |
As a result show, YC01 zymotic fluids reach 77.8% to the prevention effect of Oil Tea Anthracnose.
Prevention effect of the bacterial strain YC01 protein crude extract administrations of embodiment 3 to Oil Tea Anthracnose
The YC01 bacterial strains that embodiment 1 is separated are transferred on fresh LB flat boards and activated, and subsequent picking colony is transferred to liquid
Body LB culture medium shaken cultivation 4d, carry out centrifugation by zymotic fluid and remove thalline, supernatant is standby.Supernatant uses saturated ammonium sulfate
Method (relative saturation degree 50%) protein precipitation composition.Albumen treating method be the same as Example 2, CK groups are set altogether, and (oil tea blade is stabbed
And be inoculated with anthrax bacteria mycelia block), Control groups (healthy Camellia Leaves piece and be not inoculated with anthrax bacteria mycelia block), treatment group (oil
After tealeaves piece is stabbed, blade is uniformly smeared with YC01 protein extract dilutions, then inoculation anthrax bacteria mycelia block is in stabbing
Place).Oil tea blade incidence is observed by day and record statistics after inoculation 5d.It the results are shown in Table 2.
Prevention effect of the bacterial strain YC01 protein extracts of table 2 to Oil Tea Anthracnose
| Handle the number of sheets | The morbidity number of sheets | The incidence of disease | Prevention effect | |
| CK | 10 | 10 | 100% | -- |
| Control | 10 | 0 | 0 | -- |
| Protein extract processing | 10 | 1 | 10% | 90% |
As a result show, YC01 zymotic fluids protein extract is to Oil Tea Anthracnose prevention effect up to 90% (Fig. 4).
Prevention effect of the bacterial strain YC01 lipopeptids crude extract of embodiment 4 to Oil Tea Anthracnose
The YC01 bacterial strains that embodiment 1 is separated are transferred on fresh LB flat boards and activated, and subsequent picking colony is transferred to liquid
Body LB culture medium shaken cultivation 4d, carry out centrifugation by zymotic fluid and remove thalline, supernatant is standby.Supernatant is using salt acid deposition
Method separates its antagonistic activity composition, supernatant is adjusted into pH to 2.0 and precipitates overnight with hydrochloric acid, then 10000r/min conditions
Lower centrifugation 20min, is precipitated, and precipitation is extracted 2 times with methanol, and 2h is extracted every time, merges methanol extract.Methanol extract subtracts
0.22 μm of filter membrane, as lipopeptide compound crude extract are crossed after pressure concentration.
Lipopeptid treating method be the same as Example 2, altogether set CK groups (oil tea blade is stabbed and is inoculated with anthrax bacteria mycelia block),
Control groups (healthy Camellia Leaves piece and be not inoculated with anthrax bacteria mycelia block), treatment group are (after oil tea blade is stabbed, with YC01 fat
Peptide extract dilution uniformly smears blade, and then inoculation anthrax bacteria mycelia block is in stabbing place).It is inoculated with after 5d and observes oil by day
Tealeaves piece incidence simultaneously records statistics.It the results are shown in Table 3.
Prevention effect of the bacterial strain YC01 lipopeptids extract of table 3 to Oil Tea Anthracnose
| Handle the number of sheets | The morbidity number of sheets | The incidence of disease | Prevention effect | |
| CK | 10 | 9 | 90% | -- |
| Control | 10 | 0 | 0 | -- |
| Lipopeptid extract-treated | 10 | 1 | 10% | 88.9% |
As a result show, in YC01 zymotic fluids lipopeptid extract to Oil Tea Anthracnose prevention effect up to 88.9% (Fig. 5)
The preparation of the bacterial strain YC01 microecological microbial agents of embodiment 5
The bacterial strain YC01 that embodiment 1 is separated is activated, LB liquid medium, 160r/min, 28 DEG C of insulating box cultures are seeded to
96h。
The preparation of YC01 liquid preparations：With the bottled 100mL LB liquid mediums of 250mL triangles, conventional sterilant, after cooling
On superclean bench, picking YC01 thalline are inoculated with fermentation triangular flask, shaken cultivation, 28 DEG C, are cultivated under the conditions of 160r/min
24h is transferred to ferment tank at once, and 28 DEG C, 96h stuck fermentations are cultivated under the conditions of 160r/min.Zymotic fluid using plastic bottle or
Polybag is dispensed.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of Hunan
<120>One plant of endophytic Bacillus subtilis YC01 and its application in preventing and treating Oil Tea Anthracnose
<130> KHP171113966.5
<160> 3
<170> PatentIn version 3.3
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<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
cagagtttga tcctggct 18
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<211> 19
<212> DNA
<213>Artificial sequence
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aggaggtgat ccagccgca 19
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<211> 1509
<212> DNA
<213>Bacillus subtilis（Bacillus subtilis）YC01
<400> 1
ggttaccttg ttacgacttc accccaatca tctgtcccac cttcggcggc tggctcctaa60
aaggttacct caccgacttc gggtgttaca aactctcgtg gtgtgacggg cggtgtgtac120
aaggcccggg aacgtattca ccgcggcatg ctgatccgcg attactagcg attccagctt180
cacgcagtcg agttgcagac tgcgatccga actgagaaca gatttgtggg attggcttaa240
cctcgcggtt tcgctgccct ttgttctgtc cattgtagca cgtgtgtagc ccaggtcata300
aggggcatga tgatttgacg tcatccccac cttcctccgg tttgtcaccg gcagtcacct360
tagagtgccc aactgaatgc tggcaactaa gatcaagggt tgcgctcgtt gcgggactta420
acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtcac tctgcccccg480
aaggggacgt cctatctcta ggattgtcag aggatgtcaa gacctggtaa ggttcttcgc540
gttgcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga600
gtttcagtct tgcgaccgta ctccccaggc ggagtgctta atgcgttagc tgcagcacta660
aggggcggaa accccctaac acttagcact catcgtttac ggcgtggact accagggtat720
ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc agttacagac cagagagtcg780
ccttcgccac tggtgttcct ccacatctct acgcatttca ccgctacacg tggaattcca840
ctctcctctt ctgcactcaa gttccccagt ttccaatgac cctccccggt tgagccgggg900
gctttcacat cagacttaag aaaccgcctg cgagcccttt acgcccaata attccggaca960
acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg gctttctggt1020
taggtaccgt caaggtgccg ccctatttga acggcacttg ttcttcccta acaacagagc1080
tttacgatcc gaaaaccttc atcactcacg cggcgttgct ccgtcagact ttcgtccatt1140
gcggaagatt ccctactgct gcctcccgta ggagtctggg ccgtgtctca gtcccagtgt1200
ggccgatcac cctctcaggt cggctacgca tcgtcgcctt ggtgagccgt tacctcacca1260
actagctaat gcgccgcggg tccatctgta agtggtagcc gaagccacct tttatgtctg1320
aaccatgcgg ttcagacaac catctggtat tagccccggt ttcccggagt tatcccagtc1380
ttacaggcag gttacccacg tgttactcac ccgtccgccg ctaacatcag ggagcaagct1440
cccatctgtc cgctcgactt gcatgtatta ggcacgccgc cagcgttcgt cctgagccag1500
gatcaaact1509
Claims (5)
1. one plant of endophytic Bacillus subtilis (Bacillus subtilis) YC01, deposit number is：CGMCC No.14186.
2. the microbial inoculum containing the endophytic Bacillus subtilis YC01 described in claim 1.
3. prepare the method for microbial inoculum described in claim 2, it is characterised in that fermentation temperature is 25-28 DEG C, and rotating speed is 160-
200r/min, fermentation time is 4-8 days, and the fermenting B. subtilis YC01 in LB culture mediums prepares obtained zymotic fluid
Into liquid preparation or solid pharmaceutical preparation.
4. the answering in preventing and treating Oil Tea Anthracnose of microbial inoculum described in bacillus subtilis YC01 described in claim 1 or claim 2
With.
5. bacillus subtilis YC01 metabolite, protein crude extract administration or lipopeptid crude extract described in claim 1 are in preventing and treating oil
Application in tea anthracnose.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109797122A (en) * | 2018-12-17 | 2019-05-24 | 安徽农业大学 | The flora of one kind production L-thiamine and its application |
| CN114921358A (en) * | 2022-02-28 | 2022-08-19 | 毕节市家乡美农业综合开发有限公司 | Bacillus altitudinis strain and application thereof |
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| CN102367429A (en) * | 2011-10-24 | 2012-03-07 | 中国烟草总公司黑龙江省公司牡丹江烟草科学研究所 | Bacillus subtilis for controlling tobacco anthracnose |
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