CN107099478A - One plant of Lycoris aurea endogenetic bacteria bulkholderia cepasea HDXY 02 and its application - Google Patents
One plant of Lycoris aurea endogenetic bacteria bulkholderia cepasea HDXY 02 and its application Download PDFInfo
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- 241000196324 Embryophyta Species 0.000 title abstract description 27
- 241000894006 Bacteria Species 0.000 title abstract description 21
- 241001654189 Lycoris aurea Species 0.000 title abstract description 11
- 201000010099 disease Diseases 0.000 claims abstract description 30
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 30
- 241000233866 Fungi Species 0.000 claims abstract description 16
- 241000813090 Rhizoctonia solani Species 0.000 claims abstract description 16
- 241001508395 Burkholderia sp. Species 0.000 claims abstract description 15
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 8
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 4
- 239000012681 biocontrol agent Substances 0.000 claims abstract description 3
- 235000007164 Oryza sativa Nutrition 0.000 claims description 15
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- 108010080698 Peptones Proteins 0.000 claims description 4
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- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims description 3
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
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- 240000008574 Capsicum frutescens Species 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
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- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000879806 Fusarium oxysporum f. sp. vasinfectum Species 0.000 description 1
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- 239000002054 inoculum Substances 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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Abstract
The invention discloses the Lycoris aurea endogenetic bacteria of one plant of antagonism various plants disease fungus, specifically related to bulkholderia cepasea (Burkholderia sp.) HDXY 02, the bacterial strain oneself be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is CGMCC No 14054, and preservation date is on April 20th, 2017.The invention also discloses above-mentioned bulkholderia cepasea (Burkholderia sp.) HDXY 02 cultural method and purposes.Bulkholderia cepasea (Burkholderia sp.) HDXY 02 that the present invention is provided is strong to the antagonism of Rhizoctonia solani Kuhn, has good inhibitory activity to a variety of disease funguses, and antimicrobial spectrum is wide, can effectively suppress mycelial growth.Present invention also offers a kind of biocontrol agent for preventing and treating farm crop fungus disease, the i.e. microbial inoculum by the inoculations of HDXY 02 in culture medium obtained by fermented and cultured.The bacterial strain can be applied to suppress the microbial bacterial agent of plant disease, with good development prospect.
Description
Technical field
The invention belongs to microorganism field, and in particular to raw bulkholderia cepasea in one plant of Lycoris aurea
(Burkholderia sp.) HDXY-02, also relates to a kind of bulkholderia cepasea HDXY- for preventing and treating fungal diseases of plants
02 cultural method and purposes.
Background technology
Rice sheath blight disease is also known as moire disease, is fallen ill by Rhizoctonia solani Kuhn (Rhizoctonia solani) infection, more in height
Occur under warm super-humid conditions.The disaster that banded sclerotial blight is caused in South Rice Region of China is particularly acute, and is the main disease in current Rice Production
One of evil, the disease can prevent paddy rice from earing, or the not plump paddy of heading is more, and grain declines again.Due to the paddy rice of the water resistant sheath and culm blight of rice
The shortage of kind, current chemical bactericide is still the effective means for preventing and treating rice sheath blight disease, and wherein jinggangmeisu and Amy is
The main agricultural chemicals of application, a large amount of of Amy be polluted using meeting to environment, and the occurrence rate of the sheath blight fungus of resistance to Amy
Improve a lot；In addition, in recent years, with jinggangmeisu continue use, field have also discovered the bacterial strain of anti-jinggangmeisu.Cause
This, finding the new measure for preventing and treating rice sheath blight disease turns into the most important thing that control of plant disease works.
As the natural member of plant microecosystem, some endogenetic bacterias can be by producing antibiotic, hydrolase etc.
Antibacterial substance suppresses pathogen, to reach the purpose of controlling plant diseases.Utilize microbial control fungal diseases of plants
Have the advantages that it is environmentally safe, to person poultry harmless, slow down the pathogen resistance to the action of a drug and produce, it has also become current controlling plant diseases
Focus.Many plants are isolated to from the crops such as cotton, paddy rice, capsicum, tomato has good prevention effect to plant disease
Endogenetic bacteria, wherein have oneself through entering the application stage, and generate certain social benefit and environmental benefit.
Early stage is separated to one plant of bulkholderia cepasea HDXY-02 with antifungal activity, the bacterium from Lycoris aurea root
Strain has favorable effect to various plants disease fungus, especially best to Rhizoctonia solani Kuhn inhibition.Burkholder
Salmonella can be prevented and treated plant disease as biocontrol microorganisms, and it can be made as biological bactericide, can partly substitute chemical pesticide,
The biocontrol microorganisms of existing several plants of Burkholderias category are committed to Environmental Protection Agency (EPA) and registered as biological pesticide.Therefore,
The category bacterial strain can be played an important role in agricultural production and environmental protection, there is good application prospect.
The content of the invention：
It is an object of the invention to provide the bulkholderia cepasea that one plant has good antagonistic activity to disease fungus
(Burkholderia sp.) HDXY-02, bulkholderia cepasea (Burkholderia sp.) the HDXY-02 bacterial strains have been protected
China Committee for Culture Collection of Microorganisms's common micro-organisms center is hidden in, deposit number is CGMCC No.14054, preservation
Date is on April 20th, 2017, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and Chinese Academy of Sciences microorganism is ground
Study carefully institute.
The biological characteristics of the bulkholderia cepasea HDXY-02 is：Gram-negative (G─), cell is quarter butt
Shape, no gemma is aerobic, and oxidase test is positive, gelatin liquefaction reacting positive, catalase reacting positive；In beef extract-peptone
Bacterium colony is circular in faint yellow on culture medium solid medium, and neat in edge surface is smooth, protuberance.
The bulkholderia cepasea HDXY-02, from the Lycoris aurea root of health, has good to rice sheath blight disease
Biocontrol effect.The 16S rDNA sequences of the strain HD XY-02 are expanded, are compared online through NCBI, with reference to physiological and biochemical test knot
Fruit determines that strain HD XY-02 is bulkholderia cepasea.
Another object of the present invention is to be the provision of a kind of bulkholderia cepasea for preventing and treating fungal diseases of plants
HDXY-02 cultural method.The step of being cultivated in the fermentation medium including bulkholderia cepasea HDXY-02.
Described bulkholderia cepasea HDXY-02 or using bulkholderia cepasea HDXY-02 as the microbial inoculum of active component
Application in production falls within protection scope of the present invention.
It is yet another object of the invention to provide a kind of method for preparing biocontrol agent, the active component of the microbial inoculum is described
Bulkholderia cepasea HDXY-02.By preparing the zymotic fluid containing bulkholderia cepasea HDXY-02 or containing primary gram of Hall
The sterile fermented supernatant fluid that moral Salmonella HDXY-02 zymotic fluid is obtained after filtering, described sterile fermented supernatant fluid uses primary
Ke Huoerde Salmonella HDXY-02 zymotic fluids pass through the supernatant that centrifuging and taking is obtained, then filter acquisition by bacterial filter.Preparation method
It is simple and easy to do, organic solvent is not used, it is environmentally safe pollution-free.
The present invention also provides applications of the above-mentioned bulkholderia cepasea HDXY-02 in Rhizoctonia solani Kuhn is suppressed.It is described
Bulkholderia cepasea HDXY-02 tunning can effectively suppress the growth of Rhizoctonia solani Kuhn mycelia.
Applications of the above-mentioned Lycoris aurea endogenetic bacteria HDXY-02 in disease fungus is suppressed, i.e. bulkholderia cepasea HDXY-
The inhibitory action of 02 pair of disease fungus also includes：Rice blast fungus, fusarium graminearum, cotton-wilt fusarium and rape sclerotium
Germ, therefore, Lycoris aurea endogenetic bacteria HDXY-02 can be applied to prevent and treat rice sheath blight disease, rice blast, gibberella saubinetii
Disease, cotton wilt and sclerotinia sclerotiorum.
A kind of bulkholderia cepasea HDXY-02 that the present invention is provided, is one plant of Lycoris aurea endogenetic bacteria, to rice banded sclerotial blight
Germ bacteriostasis is strong.Strain HD XY-02 has bacteriostasis to a variety of disease funguses, with antimicrobial spectrum widely.On
State strain HD XY-02 condition of culture simple, it is easy to preserve and produce, fast growth is played in biocontrol of plant disease
Very important effect, the particularly preventing and treating to Rhizoctonia solani Kuhn have great importance, and can have as one kind and well open
Send out the broad spectrum type bactericide of application prospect.
Brief description of the drawings：
Fig. 1 is bulkholderia cepasea HDXY-02 colonial morphology.
Fig. 2 is the electrophoresis pattern of bulkholderia cepasea HDXY-02 16S rDNA sequences.
Fig. 3 is antagonism result figures of the bulkholderia cepasea HDXY-02 to Rhizoctonia solani Kuhn.
Fig. 4 is influence of the sterile fermented supernatant fluids of bulkholderia cepasea HDXY-02 to Rhizoctonia solani Kuhn mycelial growth.
Embodiment：
With reference to embodiments and accompanying drawing, the embodiment to the present invention is described in further detail.
Culture medium prescription used in following examples is：
PDA solid culture based formulas：Potato 200g, sucrose 20g, agar 20g, distilled water are settled to 1L, and pH is natural；
NA culture medium prescriptions：Beef extract 3g, peptone 10g, sodium chloride 5g, agar 20g, distilled water is settled to 1L, pH
7.3；
NB culture medium prescriptions：Beef extract 3g, peptone 10g, sodium chloride 5g, distilled water are settled to 1L, pH 7.3.
Embodiment 1：The separation of endophyte
Take Lycoris aurea root appropriate, soak 1min with 70% ethanol on superclean bench, then soaked with 0.1% mercuric chloride
6min, optionally soaks 1min using 70% ethanol, uses aseptic water washing 3-5 times.The root for weighing 1g surface sterilizings is dipped in
Ground in 10mL water in sterile mortar, take supernatant to do gradient dilution with sterilized water after standing, take appropriate dilution factor in NA
Even spread on flat board, is inverted after culture, 2-3d after culture dish is dry at 28 DEG C, by the bacterium colony of different size, color and form
Line purifying is carried out on picking to new NA flat boards, the single bacterium colony to be measured being separated to is numbered, and will with glycerol stocks method
Purified bacterium colony is placed in standby in ultra low temperature freezer.
Embodiment 2：Antagonisms of the strain HD XY-02 to a variety of disease funguses
The endogenetic bacteria that the activation of NA culture mediums is separated to, carries out antagonistic effect, using PDA plate culture medium, using flat board
Face-off method, aseptic card punch, which is beaten, takes a diameter of 6mm's to supply examination fungi bacteria cake, is placed in a diameter of 90mm PDA plate center, culture
After 2-3d, the endophyte of activation is inoculated in the both sides away from fungus block 3cm, 28 DEG C incubated 3-5 days.Only to connect pathogen, no
It is control to connect isolated strains, and timing is observed, antibacterial bandwidth and colony radius between measuring endophyte to supplying examination disease fungus,
It is used as the index for judging its antagonistic ability.Obtain one plant of endophyte HDXY-02 to various plants pathogen with antagonism.
Fig. 3 is antagonism design sketch of the strain HD XY-02 to Rhizoctonia solani Kuhn.
Result of the test shows that bulkholderia cepasea HDXY-02 of the present invention is to Rhizoctonia solani Kuhn (Rhizoctorzia
Solani), fusarium graminearum (Fusariumt graminearumt), rice blast fungus (Magnaporthe
Oryzae), Sclerotinia sclerotiorum (Sclerotinia libertiana) and cotton-wilt fusarium (Fusarium oxysporum
F.sp.vasinfectum) this 5 kinds of disease funguses are respectively provided with bacteriostasis.As a result as shown in table 1, especially to rice sheath blight disease
The inhibition of bacterium is optimal, and inhibiting rate reaches more than 72%, therefore strain HD XY-02 is one plant and disease fungus is pressed down with relatively strong
The bacterial strain of making.
The strain HD XY-02 of table 1 and various plants disease fungus dual test result
Embodiment 3：The clonal analysis of strain HD XY-02 of the present invention 16S rDNA sequences
Strain HD XY-02 genomic DNAs are extracted, in this, as template, its 16S is expanded using bacterial universal primers PCR
RDNA fragments.Take PCR primer to carry out after 1% agarose gel electrophoresis detection, by PCR primer recovery purifying, serve Hai Shenggong
Sequencing.PCR the primers are：16S-27：5’-AGAGTTTGATCCTGGCTCAG-3’；16S-1492:5’-
GGTTACCTTGTTACGACTT-3’.PCR reaction systems (25 μ L)：Dilution DNA template l μ L；dNTP 2μL；Primer 16S-27l μ
L；Primer 16S-1492l μ L；10×PCR Buffer 2.5μL；The μ L of Taq archaeal dna polymerases 0.2；Sterilizing ultra-pure water complements to end
The μ L of volume 25.PCR reaction conditions are 94 DEG C of 5min；94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 1.5min, 28 circulations；72℃10min.
16S rDNA sequences (shown in SEQ ID No.1) BLAST analysis results show bacterial strain of the present invention and Burkholder
Salmonella (Burkholderia sp.) homology reaches 99%.Colonial morphology, physiological and biochemical test result and 16S with reference to bacterial strain
RDNA sequence analyses, bulkholderia cepasea (Burkholderia sp.) is accredited as by strain HD XY-02.
Embodiment 4：Strain HD XY-02 cultural method
Comprise the following steps that：Activate 22-26h in A, picking strain HD XY-02 to 5mL NB nutrient solutions, 28-30 DEG C of temperature,
Shaking speed 180-220r/min；
B, the strain HD XY-02 nutrient solutions of activation in step (A) are drawn into 1mL (inoculum concentration 2%) be inoculated into containing 50mL
Fermentation medium (fermentative medium formula：Peptone 10-20g, glycerine 5-15mL, dipotassium hydrogen phosphate 1-3g, magnesium sulfate 0.5-
2g, is settled to 1L, pH6.5-7.5) 250mL triangular flasks in, 28-30 DEG C of temperature, shaking speed 180-220r/min, concussion training
Support 48h-72h；
C, the zymotic fluid for obtaining step (B) 5000-8000rpm under the conditions of 4 DEG C centrifuge 10-20min, then filtered through bacterium
Device (0.22 μm) is filtrated to get sterile fermented supernatant fluid；
D, with sterilized water thalline is washed 3-5 times, its bacteria suspension is made.Sterile fermentation supernatant, bacteria suspension are placed in 4 DEG C of bars
Saved backup under part.
Embodiment 5：Inhibitory action of the strain HD XY-02 without fermented liquid to Rhizoctonia solani Kuhn mycelial growth
A, strain HD XY-02 is seeded in 5mL NB nutrient solutions activates 24h, the HDXY-02 1mL of activation are inoculated into
In 250m1 triangular flasks containing 50mL fermentation mediums, 28 DEG C, 200rpm, shaken cultivation 48h.Zymotic fluid is put 4 DEG C,
8000rpm centrifuges 10min, and supernatant (0.22 μm) filtering of bacterial filter obtains without fermented liquid.
B, fermented supernatant fluid is added into PDA solids in 1%, 5%, 10%, 15%, 20%, 25% and 30% ratio trained
Support in base, a diameter of 5mm bacterium mycelia block be inoculated with each flat board, each 3 repetitions of processing, take 3 parallel tests to obtain result,
28 DEG C culture, in after 5d measure Rhizoctonia solani Kuhn colony diameter and record related data.
Result of the test is shown in Fig. 4, and strain HD XY-02 various concentrations without fermented liquid can suppress Rhizoctonia solani Kuhn mycelia
Growth, and gradually strengthen with the increase inhibiting rate of concentration.The sterile of strain HD XY-02 various concentrations is observed on PDA plate
Ferment filtrate is suppressed to the visible mycelial growth of inhibition of Rhizoctonia solani Kuhn mycelial growth, slow-growing phenomenon.
SEQUENCE LISTING
<110>Institute of Botany
<120>One plant of Lycoris aurea endogenetic bacteria bulkholderia cepasea HDXY-02 and its application
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1437
<212> DNA
<213>Bulkholderia cepasea（Burkholderia sp.）HDXY-02
<400> 1
attggcggct gcctttccat gcaagtcgaa cggcagcacg ggtgcttgca cctggtggcg 60
agtggcgaac gggtgagtaa tacatcggaa catgtcctgt agtgggggat agcccggcga 120
aagccggatt aataccgcat acgatctacg gatgaaagcg ggggaccttc gggcctcgcg 180
ctatagggtt ggccgatggc tgattagcta gttggtgggg taaaggccca ccaaggcgac 240
gatcagtagc tggtctgaga ggacgaccag ccacactggg actgagacac ggcccagact 300
cctacgggag gcagcagtgg ggaattttgg acaatgggcg aaagcctgat ccagcaatgc 360
cgcgtgtgtg aagaaggcct tcgggttgta aagcactttt gtccggaaag aaatcctgag 420
ggctaatatc cttcggggat gacggtaccg gaagaataag caccggctaa ctacgtgcca 480
gcagccgcgg taatacgtag ggtgcgagcg ttaatcggaa ttactgggcg taaagcgtgc 540
gcaggcggtt tgttaagacc gatgtgaaat ccccgggctc aacctgggaa ctgcattggt 600
gactggcaag ctagagtatg gcagaggggg gtagaattcc acgtgtagca gtgaaatgcg 660
tagagatgtg gaggaatacc gatggcgaag gcagccccct gggccaatac tgacgctcat 720
gcacgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc cctaaacgat 780
gtcaactagt tgttggggat tcatttcctt agtaacgtag ctaacgcgtg aagttgaccg 840
cctggggagt acggtcgcaa gattaaaact caaaggaatt gacggggacc cgcacaagcg 900
gtggatgatg tggattaatt cgatgcaacg cgaaaaacct tacctaccct tgacatggtc 960
ggaaccttgg agagatccga gggtgctcga aagagaaccg atacacaggt gctgcatggc 1020
tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgtc 1080
cttagttgct acgcaagagc actctaggga gactgccggt gacaaaccgg aggaaggtgg 1140
ggatgacgtc aagtcctcat ggcccttatg ggtagggctt cacacgtcat acaatggtcg 1200
gaacagaggg tcgccaaccc gcgaggggga gctaatccca gaaaaccgat cgtagtccgg 1260
attgcactct gcaactcgag tgcatgaagc tggaatcgct agtaatcgcg gatcagcatg 1320
ccgcggtgaa tacgttcccg ggtcttgtac acaccgcccg tcacaccatg ggagtgggtt 1380
ttaccagaag tggctagtct aaccgcaagg aggacggtca cccacggtag gatctac 1437
Claims (6)
- Bulkholderia cepasea 1. (Burkholderia sp.) HDXY-02, the strain was preserved in China on April 20th, 2017 Microbiological Culture Collection administration committee common micro-organisms center, its deposit number is：CGMCC No.14054.
- 2. the strain culturing that bulkholderia cepasea (Burkholderia sp.) HDXY-02 as claimed in claim 1 is obtained Thing, fermentation culture or zymotic fluid sterile supernatant.
- 3. bulkholderia cepasea (Burkholderia sp.) HDXY-02 described in claim 1 is the biology of active component Microbial inoculum, it is characterised in that the biocontrol agent is prepared using following methods：By the bulkholderia cepasea described in claim 1 (Burkholderia sp.) HDXY-02 seed liquor is inoculated in culture medium, 28-30 DEG C of temperature, shaking speed 180-220r/ Min, concussion and cultivate 48h-72h；The formula of wherein culture medium is：Peptone 10-20g, glycerine 5-15mL, dipotassium hydrogen phosphate 1- 3g, magnesium sulfate 0.5-2g, are settled to 1L, pH6.5-7.5.
- 4. bulkholderia cepasea (Burkholderia sp.) HDXY-02 described in claim 1 is in antagonizing pathogenic fungi Application.
- 5. the culture of bulkholderia cepasea (Burkholderia sp.) HDXY-02 described in claim 2, fermented and cultured Liquid or zymotic fluid sterile supernatant are applied in antagonizing pathogenic fungi.
- 6. the application described in claim 4 or 5, it is characterized in that the disease fungus is Rhizoctonia solani Kuhn, wheat scab, water Rice Pyricularia oryzae, cotton-wilt fusarium and Sclerotinia sclerotiorum.
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