CN106497852A - A kind of bulkholderia cepasea bacterial strain ES 89 of preventing and treating fungal diseases of plants and cultural method and application - Google Patents
A kind of bulkholderia cepasea bacterial strain ES 89 of preventing and treating fungal diseases of plants and cultural method and application Download PDFInfo
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- CN106497852A CN106497852A CN201611197281.XA CN201611197281A CN106497852A CN 106497852 A CN106497852 A CN 106497852A CN 201611197281 A CN201611197281 A CN 201611197281A CN 106497852 A CN106497852 A CN 106497852A
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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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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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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
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses a kind of bulkholderia cepasea bacterial strain ES 89 of preventing and treating fungal diseases of plants and cultural method and application, step is：A, by 89 strains of ES be inoculated in nutrient broth culture fluid activate；B, 89 strains of ES of activation are inoculated in the triangular flask containing nutrient broth culture fluid, shaken cultivation；C, by shake training after obtain fermentation liquid be placed under certain condition be centrifuged, biofilter filter high fermentation liquid, obtain aseptic supernatant；D, thalline is washed with sterilized water, make thalline suspension, fermentation liquid, fermented supernatant fluid, thalline suspension are preserved.The bacterial strain preservation CCTCC, CCTCC NO：The application of M 2016665, bacterial strain ES 89 in treatment or prevention plant pathogenic fungi medicine is prepared.The bacterial strain is inhibited to Rhizoma Coptidis leaf spot fungi etc., can be used to prevent and treat plurality of plant diseases.The bacterial strain reaches 88 more than % to Rhizoma Coptidis leaf spot prevention effect, easy to implement the method, easy to operate, reduces the pollution in production process, will not produce the poisoning of solvent, and application prospect is preferable.
Description
Technical field
The invention belongs to technical field of plant disease biological control, and in particular to a kind of Bai Ke of preventing and treating fungal diseases of plants
Hall moral Salmonella (Burkholderia sp.) bacterial strain ES-89, also relates to a kind of Bai Kehuo of preventing and treating fungal diseases of plants
The cultural method of your moral Salmonella bacterial strain ES-89, further relates to a kind of bulkholderia cepasea bacterial strain ES- of preventing and treating fungal diseases of plants
89 purposes.
Background technology
Rhizoma Coptidis are a kind of conventional Chinese crude drugs, in China river Hubei Province Hunan and Guizhou mountain region, river Southwest Mountainous Areas, river Yunnan-Tibet South Mountain ground, the Qin
The areas such as Bashan Mountain ground plantation.Coptis medicinal is worth height, has been reported in the aspects such as anticancer and blood sugar lowering with good therapeutic effect, has drawn
People's extensive concern is played.As international community is continuously increased to natural drug exploitation temperature, especially Japan is to coptis detoxifcation
The further investigation and extensive application of soup, China's Coptis planting and export volume increase year by year.In recent years, disease problem is to affect Huang
The important restriction factor that adhesion is produced；In Hubei Province, Enshi City investigation finds, (pathogen is Aquilegia viridiflora Phoma sp to Rhizoma Coptidis leaf spot
Phoma aquilegiicola) occur universal, general field sickness rate is reached when occurring serious between 10～30%
100%, Rhizoma Coptidis yield is had a strong impact on, administration agriculture brings huge economic loss.
The at present preventing and treating of Rhizoma Coptidis leaf spot due to unreasonable use chemical pesticide for a long time, is given mainly based on chemical prevention
Ecological environment and human health bring very important harm；Another aspect pathogen to commonly use chemical agent Drug resistance by
Cumulative strong so that preventive effect constantly declines.And Biological control is safe to environment, ecological and human health because having, obtain
The extensive attention of people has simultaneously become study hotspot.We are separated from the Rhizoma Coptidis rhizosphere soil from enshi early stage
To one plant of biocontrol microorganisms bulkholderia cepasea ES-89 for having an antifungal activity, the bacterial strain various plants pathogenic fungi is had compared with
Good preventive effect, especially best to Rhizoma Coptidis leaf spot preventive effect.Since late 1980s, existing several plants of Burkholderias category
Biocontrol microorganisms are delivered to U.S. Environmental Protection Agency (EPA) (EPA), and registration is used as biological pesticide in succession, but yet there are no Burkholder at home
Salmonella prevents and treats the report of Rhizoma Coptidis disease.
Content of the invention
The purpose of the present invention is to there are provided a kind of bulkholderia cepasea bacterial strain ES- of preventing and treating fungal diseases of plants
89, the tunning of the bacterial strain is distorted can Rhizoma Coptidis leaf spot fungi mycelia, and effectively inhibits mycelial growth, so as to reach
The purpose that big Tanaka prevents and treats Rhizoma Coptidis leaf spot is arrived.The bacterial strain delivers to China typical culture collection on November 23rd, 2016
Center preservation, Classification And Nomenclature：Burkholderia sp.ES-89；Deposit number：CCTCC NO：M 2016665；Address：China
Wuhan Wuhan University.
Another object of the present invention is to there are provided a kind of bulkholderia cepasea bacterium of preventing and treating fungal diseases of plants
The cultural method of strain ES-89, the biocontrol agent prepared by the bacterium contain bulkholderia cepasea ES-89 or containing primary gram of Hall
The bacteria-free filtrate that moral Salmonella ES-89 fermentation liquids are obtained after filtering, described bacteria-free filtrate use bulkholderia cepasea ES-89
Fermentation liquid is filtered through biofilter through the supernatant that centrifuging and taking is obtained again and is obtained.Easy to implement the method, easy to operate, and in producing
Do not use organic solvent, reduce in production process and product use in pollution, will not also produce the poisoning of solvent.
A further object of the invention is to there are provided a kind of bulkholderia cepasea bacterium of preventing and treating fungal diseases of plants
Applications of the strain ES-89 in the plant disease medicine that treatment or plant pathogenic fungi cause is prepared.The plant pathogenic fungi is Huang
Connect Leaf blotch pathogeny Aquilegia viridiflora Phoma sp (Phoma aquilegiicola), with good prevention effect.
To achieve the above object, the present invention adopts following technical measures：
A kind of bulkholderia cepasea bacterial strain ES-89, is obtained in the following manner：Microorganism used therefor of the present invention is in Hubei
Separate in the Rhizoma Coptidis rhizosphere soil of province Enshi Taishan mausoleum Rhizoma Coptidis GAP planting bases, one plant obtained is screened to Rhizoma Coptidis leaf spot
There is the bacterial strain of stronger prevention effect, by morphology and the identification of molecular biology, be accredited as bulkholderia cepasea
(Burkholderia sp.), applicant is named as ES-89, and it is military that the bacterial strain delivered Hubei Province on November 23rd, 2016
Chinese Typical Representative culture in Wuhan University of Chinese city preserves center (CCTCC) preservation, and its preserving number is CCTCC NO：M
2016665.
The mycology feature of bulkholderia cepasea (Burkholderia sp.) ES-89 thalline：Bulkholderia cepasea ES-
89 in Nutrient agar (NA) culture medium bacterium colony be milky, neat in edge, subcircular, smooth surface swell, opaque, in oil
Shape, with the growth of incubation time, thalline culture is more sticky.Cell is oval to circle, 0.5-0.6 × 1-2 μm, gram
Staining reaction is redness, is gram negative bacteria.
A kind of cultural method of the bulkholderia cepasea bacterial strain ES-89 of preventing and treating fungal diseases of plants, its step are as follows：
A, ES-89 strain 1ml are inoculated in 100ml nutrient broths (NB) culture fluid activation 22-26h；
B, the ES-89 strain 1ml of activation in step (A) are inoculated into containing 100ml nutrient broths (NB) culture fluid
In 250ml triangular flasks, 26-30 DEG C, 160rpm, shaken cultivation 46-50h；
C, by shake in step (B) training after obtain fermentation liquid be placed in 4 DEG C under the conditions of, 8000rpm centrifugation 18-22min, carefully
(0.22 μm) filtration high fermentation liquid of bacterium filter, obtains aseptic fermented supernatant fluid；
D, thalline is washed 2-4 time with sterilized water, make its thalline suspension.Fermentation liquid, fermented supernatant fluid, thalline are hanged
Liquid is preserved under the conditions of being placed in 4 DEG C, standby；
Described nutrient broth (NB) culture fluid component and formula are：Beef extract 5g, peptone 10g, sucrose 10g, mend
Distilled water is filled to 1000ml, adjust pH to 7.2-7.4, in 121 DEG C of high pressure steam sterilization 30min.
Nutrient agar (NA) nutrient media components and formula are：Beef extract 5g, peptone 10g, sucrose 10g, add agar
15-20g, supplements distilled water to 1000ml, adjusts pH to 7.2-7.4, in 121 DEG C of high pressure steam sterilization 28-32min.
Potato dextrose agar (PDA) nutrient media components and formula are：Rhizoma Solani tuber osi 200g, glucose 20g, add agar
15-20g, supplements distilled water to 1000ml, and natural pH, in 121 DEG C of high pressure steam sterilization 30min.
A kind of bulkholderia cepasea bacterial strain ES-89, the bacterial strain contain its sequence for the nucleotide shown in SEQ ID NO.1
Sequence.The preservation of bulkholderia cepasea ES-89 bacterial strain prepared by the present invention：The thalline of bulkholderia cepasea is stored in
In 25% glycerol, -20 DEG C of freezings.
A kind of bulkholderia cepasea bacterial strain ES-89 of preventing and treating fungal diseases of plants is preparing treatment or prevention pathogenic
Application in fungi-medicine, including being prepared into the medicine of preventing and treating Rhizoma Coptidis leaf spot by the use of the bacterial strain as main component, described
Bulkholderia cepasea bacterial strain ES-89 is particularly well-suited to answering in the medicine for prevent and treat Rhizoma Coptidis Leaf blotch pathogeny Aquilegia viridiflora Phoma sp
With.Its step is：
A, a kind of bulkholderia cepasea bacterial strain ES-89 and Rhizoma Coptidis Leaf blotch pathogeny flat board dual test；
A kind of inhibitory action of B, bulkholderia cepasea bacterial strain ES-89 tunnings to Rhizoma Coptidis leaf spot fungi mycelial growth
Impact with mycelial structure；
C, a kind of bulkholderia cepasea bacterial strain ES-89 tunnings are ground in vitro prevention effect in Rhizoma Coptidis leaf spot room
Study carefully；
A kind of research of D, bulkholderia cepasea bacterial strain ES-89 tunnings to the potted plant prevention effect of Rhizoma Coptidis leaf spot.
Described plant pathogenic fungi is specially Rhizoma Coptidis leaf spot fungi (Phoma aquilegiicola), grape ulcer
Pathogenic bacteria (Botryosphaeria dothidea), tobacco brown spot pathogen (Alternaria alternata), Pyricularia oryzae
(Magnaporthe oryzae), Flos Ilicis Asprellae anthrax bacteria (Colletotrichum higginsianum).
The present invention compared with prior art, with advantages below and effect：
1st, bulkholderia cepasea ES-89 is the biocontrol bacterial strain being separated to from Rhizoma Coptidis root soil, to Rhizoma Coptidis leaf spot
Preventive effect preferably, and is processed Rhizoma Coptidis plant and can reach environmental protection, safe efficient using the tunning of bulkholderia cepasea ES-89
Purpose.
2nd, at present, the relevant report of Rhizoma Coptidis biological control of diseases medicament, the Burkholderia in the present invention yet there are no
Bacterium ES-89 can provide strain resource for the production of Rhizoma Coptidis disease biological and ecological methods to prevent plant disease, pests, and erosion medicament.
3rd, bulkholderia cepasea ES-89 antibacterial band mean breadth to Rhizoma Coptidis leaf spot fungi is found in flat board face-off experiment
1.9cm is reached, average bacteriostasis rate is more than 68%；ES-89 variable concentrations tunning can suppress Rhizoma Coptidis leaf spot fungi bacterium
Silk growth, and gradually strengthen with the increase suppression ratio of concentration, in the potato glucose fine jade containing 10% (V/V) fermented supernatant fluid
On fat (PDA) flat board, its inhibition percentage reaches more than 52%；When fermented supernatant fluid and PDA volume ratios are 25%, which suppresses
Percentage rate has reached more than 95%.
4th, excised leaf test shows, after ES-89 fermentation liquor treatments, scab mean size is only 0.32cm, relative to
The 1.5-2.1cm Lesion sizes of matched group, its suppression ratio have reached more than 80%.
5th, potted plant preventing and treating experiment shows that the Rhizoma Coptidis blade state of an illness of bulkholderia cepasea fermentation liquor treatment is lighter, and which is fallen ill
Rate is only 12.5%, and average disease index is 3.1, and prevention effect reaches more than 88%.
6th, the bulkholderia cepasea that the present invention is used is a kind of good biocontrol bacterial strain of preventing and treating Rhizoma Coptidis leaf spot effect, can
Biological control for phytopathogen；The features such as there is green safety, good environment compatibility, low production cost.
7th, the present invention directly prevents and treats Rhizoma Coptidis leaf spot using the fermentation liquid of bulkholderia cepasea ES-89 bacterial strain, produce with
And be suitable for all very simples, do not use organic solvent in production, reduce in production process and product use in pollution,
The poisoning of solvent will not be also produced, with very wide application prospect.
Description of the drawings
Fig. 1 is a kind of colonial morphology of the bulkholderia cepasea bacterial strain ES-89 of preventing and treating fungal diseases of plants.
Fig. 2 is a kind of thalline microscopic morphology of the bulkholderia cepasea bacterial strain ES-89 of preventing and treating fungal diseases of plants.
Fig. 3 is a kind of rDNA-ITS areas amplification sequence of the bulkholderia cepasea bacterial strain ES-89 of preventing and treating fungal diseases of plants
Row electrophoretogram.
Fig. 4 be a kind of preventing and treating fungal diseases of plants bulkholderia cepasea bacterial strain ES-89 to Rhizoma Coptidis Leaf blotch pathogeny
Antagonism and control.
Wherein：Fig. 4 A show antagonisms of the ES-89 to Rhizoma Coptidis leaf spot fungi in PDA culture medium, and Fig. 4 B are controls
(not being inoculated with biocontrol bacterial strain ES-89).
Fig. 5 be a kind of preventing and treating fungal diseases of plants bulkholderia cepasea bacterial strain ES-89 tunnings to Rhizoma Coptidis tikka
The impact (block diagram) of pathogenic bacteria mycelial growth.
Fig. 6 be a kind of preventing and treating fungal diseases of plants bulkholderia cepasea bacterial strain ES-89 tunnings to Rhizoma Coptidis tikka
Pathogenic bacteria mycelial growth inhibitory action.
Wherein：6A is control, and 6B is the histamine result that Fermentation Substance Concentration is 5%, and it is 15% that 6C is Fermentation Substance Concentration
Histamine result, 6D is the histamine result that Fermentation Substance Concentration is 25%.
Fig. 7 be a kind of preventing and treating fungal diseases of plants bulkholderia cepasea bacterial strain ES-89 tunnings to Rhizoma Coptidis tikka
Result figure under 40 times of mirrors of impact of pathogenic bacteria mycelial structure.
Wherein：Fig. 7 A, 7B show the micro- of Rhizoma Coptidis leaf spot fungi mycelia in the PDA culture medium containing ES-89 tunnings
Form, Fig. 7 C are controls (PDA culture medium without ES-89 tunnings).
Fig. 8 be a kind of preventing and treating fungal diseases of plants bulkholderia cepasea bacterial strain ES-89 fermentation liquids to Rhizoma Coptidis leaf spot
Inhibitory action.
Wherein：8A is compareed (without ES-89 fermentation liquor treatments, aseptic water process), and 8B, 8C show ES-89 fermentation liquids
Inhibitory action to Rhizoma Coptidis leaf spot.
Fig. 9 be a kind of preventing and treating fungal diseases of plants bulkholderia cepasea bacterial strain ES-89 fermentation liquids to Rhizoma Coptidis leaf spot
Potted plant preventive effect.Wherein：9A is negative control (without ES-89 fermentation liquor treatments, aseptic water process), and 9B is fermented through ES-89
The Rhizoma Coptidis incidence of liquid process, 9C is positive control (process of 10% 1500 times of Difenoconazole liquid).
Specific embodiment
Technical scheme of the present invention, is such as not specifically noted, and is the ordinary skill in the art.
Embodiment 1：The screening of bulkholderia cepasea bacterial strain ES-89
The screening of bacterial strain：
(1) collection of sample：This experiment soil picks up from Hubei China province Enshi Taishan mausoleum Rhizoma Coptidis GAP planting bases, adopts
It is sampled with five point sampling, around Rhizoma Coptidis rhizosphere and underground 15 or 17 or 19 or 20cm, per takes for the depth of sampling
Sample 200 or 240 or 260 or 280 or 300g, taking back laboratory in the sealed bag that then sample is loaded sterilizing carries out the sieve of antibacterial
Choosing.
(2) preparation of sample suspension：5g will be taken after soil sample grinding (cross 80 mesh sieves) will be collected is added to that to fill 45ml aseptic
In the triangular flask of water, then 28 DEG C of shaking table 180rpm fully shakings 20min make 1:The Soil Slurry of 10 (V/V).Treat grogs
After staticly settling, 1ml supernatant is drawn, move in the test tube for filling 9ml sterilized water, make 1:The soil of 100 (V/V) concentration hangs
Liquid, by that analogy, makes 1:1000(V/V)、1:The variable concentrations suspensions such as 10000 (V/V).
(3) separation of antibacterial：Draw 10-4、10-5With 10-6The each 0.2ml of the suspension of gradient is in NA (beef extract 5g, egg
White peptone 10g, sucrose 10g, add agar 15-20g, distilled water 1000ml) even spread on flat board, after culture dish natural air drying
Culture is inverted at 28 DEG C, starting picking single bacterium colony after 24h carries out purification culture on new NA flat boards, to treating for being totally separated
Survey bacterial strain to be numbered, then its antagonistic activity is screened.
(4) screening of biocontrol bacterial strain：Using traditional 5 points of face-offs plating methods, detect above-mentioned be separated to various treat
Survey bacteriostatic activity of the bacterial strain to Rhizoma Coptidis leaf spot fungi.25 DEG C of cultures pathogen of 3 days is cut with 6mm card punch and is inoculated into Rhizoma Solani tuber osi
Agar glucose (PDA) plate center, while 4 test strains of equidistant inoculation on the circumference of radius 3cm, compare as not connecing
Plant test strains.Cultivate at 25 DEG C, day by day observed and recorded, and measure the antibacterial band between test strains and Rhizoma Coptidis leaf spot fungi
Width, used as the strong and weak index of antagonistic activity.Through above-mentioned screening, obtain one plant and there is to Rhizoma Coptidis leaf spot strong inhibition to act on
Bacterial strain, numbering is ES-89, saves backup.
Embodiment 2：The identification of bulkholderia cepasea bacterial strain ES-89
The identification of bacterial strain：
Applicant is separated from Chinese Enshi State of Hubei Province Taishan mausoleum Rhizoma Coptidis GAP planting bases Rhizoma Coptidis rhizosphere soil, screening
One plant of bulkholderia cepasea bacterial strain to Rhizoma Coptidis leaf spot with stronger prevention effect is obtained, applicant is named as ES-
89.The separation of the ES-89 bacterial strains of the present invention and authentication method, with reference to plant pathology research method (Fang Zhongda, 1998), uncle outstanding thin
2001) dientification of bacteria handbook (the 8th edition) and common bacteria system identification handbook (east show pearl and Cai Miaoying, the test side in monograph such as
Method.ES-89 bacterial strains are placed in NA cultures based on 28 DEG C of incubated overnight, using bacterial genomes DNA extraction kit (TIANGEN)
Genomic DNA is extracted, using amplification bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1494R (5'-
TACGGYTACCTTGTTACGAC TT-3') its 16S rDNA area is expanded.
ES-89 strain morphology qualification results：Colony characteristicses such as Fig. 1 shows, Initial stage of culture colonial morphology in NA culture medium
Smooth, milky, bacterium colony are opaque, and after culture 48h, bacterium colony presentation is faint yellow, and with the prolongation of incubation time, bacterium colony becomes oil
Stain shape, in expansion process, colony edge merges to form oily, and half glue is liquid；Positive and negative color is identical, is combined loosely with culture medium
Gu.Microscope inspection feature Fig. 2 shows that ES-89 strain cells are oval to circle, 0.5-0.6 × 1-2um；Gram’s staining is anti-
Should be red, be gram negative bacteria.
Sequencing obtains the DNA fragmentation (Fig. 3) of 1431bp sizes, and sequence is in NCBI websites (http://
Www.ncbi.nlm.nih.gov) BLAST analyses are carried out, it is known which is high with bulkholderia cepasea (Burkholderia sp.)
Degree is homologous, and homology reaches 100%.During the bacterial strain is delivered in the Wuhan University of Wuhan City, Hubei Province on November 23rd, 2016
State's Culture Collection (CCTCC) preservation, its preserving number are CCTCC NO：M 2016665.A kind of Burkholderia
Bacteria strain ES-89, the bacterial strain contain its sequence for the nucleotide sequence shown in SEQ ID NO.1.Find in flat board face-off experiment
Bulkholderia cepasea is to the average bacteriostasis rate of Rhizoma Coptidis Leaf blotch pathogeny more than 68%；The fermentation of biocontrol bacterial strain variable concentrations is produced
Thing can suppress Rhizoma Coptidis leaf spot fungi mycelial growth, and gradually strengthen with the increase suppression ratio of concentration, when containing 25% (V/
V) on the PDA plate of fermented supernatant fluid, its inhibition percentage has reached more than 95%；Potted plant preventing and treating experiment show, primary gram of Hall
The Rhizoma Coptidis blade state of an illness of moral Salmonella fermentation liquor treatment is lighter, and its sickness rate is only 12.5%, and average disease index is 3.1, preventing and treating
Effect reaches more than 88%；Therefore bulkholderia cepasea is one plant of preventing and treating good biocontrol bacterial strain of Rhizoma Coptidis leaf spot effect, can
Biological control for phytopathogen.And the bacterial strain has, and green safety, environment compatibility be good, the low spy of production cost
Point, with very wide application prospect.
Embodiment 3：
A kind of bulkholderia cepasea ES-89 cultural method of preventing and treating fungal diseases of plants, its step are as follows：
A, by ES-89 strain 1ml be inoculated in 100ml NB culture fluid activate 22 or 23 or 24 or 25 or 26h；
B, the ES-89 strain 1ml of activation in step (A) are inoculated into the 250ml triangular flasks containing 100ml NB culture fluid
In, 26 or 27 or 28 or 29 or 30 DEG C, 160rpm, shaken cultivation 46 or 47 or 48 or 49 or 50h；
C, by shake in step (B) training after obtain fermentation liquid be placed in 4 DEG C under the conditions of, 8000rpm centrifugation 18 or 19 or 20 or
21 or 22min, biofilter (0.22um) filters high fermentation liquid, obtains aseptic fermented supernatant fluid；
D, thalline is washed 2 or 3 or 4 times with sterilized water, make its thalline suspension.Fermentation supernatant, thalline suspension are placed in 4
Save backup under the conditions of DEG C；
Described nutrient broth (NB) culture fluid component and formula are：Beef extract 5g, peptone 10g, sucrose 10g, mend
Distilled water is filled to 1000ml, adjust pH to 7.2-7.4, in 121 DEG C of high pressure steam sterilization 30min.
Nutrient agar (NA) nutrient media components and formula are：Beef extract 5g, peptone 10g, sucrose 10g, add agar
15-20g, supplements distilled water to 1000ml, adjusts pH to 7.2-7.4, in 121 DEG C of high pressure steam sterilization 28-32min.
Potato dextrose agar (PDA) nutrient media components and formula are：Rhizoma Solani tuber osi 200g, glucose 20g, add agar
15-20g, supplements distilled water to 1000ml, and natural pH, in 121 DEG C of high pressure steam sterilization 30min.
In this cultural method, the time of bacterial strain uses therefor activation is 24h or so, during using shaken cultivation, when strain culturing 24h
When its growth rate reach maximum, activity is most strong；When strain culturing 48h or so, its growth population reaches peak value, therefore now
The fermentation liquid effect for obtaining is best；4 DEG C, under the conditions of 8000rpm centrifugation not only can keep ferment product to the full extent
Activity, can also preferably isolate the fermented supernatant fluid and bacterial strain thalline of bacterial strain, be easy to subsequent experimental；Obtained by this experiment
The aseptic fermented supernatant fluid of bulkholderia cepasea ES-89, thalline suspension and tunning, more directly can study
The fermenting mechanism of the bacterial strain, the resisting substance or thalline during further the antagonism of the clearly bacterial strain is by fermented supernatant fluid
In antibacterial substance determined.
Embodiment 4：ES-89 bacterial strains and the flat board dual test of various plants pathogenic fungi
A small amount of ES-89 bacterial strains are picked with inoculating loop to rule in the middle of PDA plate, replaces with sterilized water ES-89 fermentation liquids to make
Control, is repeated 3 times, and places superclean bench and dries up.Beaten with the card punch of internal diameter 6mm and take the Rhizoma Coptidis leaf spot fungi for having activated
(Phoma aquilegiicola), grape ulcer pathogenic bacteria (Botryosphaeria dothidea), tobacco brown spot pathogen
(Alternaria alternata), Pyricularia oryzae (Magnaporthe oryzae), Flos Ilicis Asprellae anthrax bacteria
(Colletotrichum higginsianum) colony edge mycelia block, is inoculated in the both sides of bacterial strain ES-89, mycelia block respectively
Between spacing be 6mm.The size of its antagonism is observed after 25 DEG C of culture 5d, and measures its antibacterial bandwidth.Mycelial growth
Suppression ratio (%)=(control colony radius-process colony radius)/control colony radius × 100%, result of the test are shown in Fig. 4 and Biao
1.
Table 1ES-89 bacterial strains and various plants pathogenic fungi flat board dual test (5d, 25 DEG C)
Dual test result is as shown in table 1：Bulkholderia cepasea ES-89 has to multiple important plant pathogenic fungis
Antagonism, especially the inhibition to Rhizoma Coptidis Leaf blotch pathogeny is optimal, and suppression ratio reaches more than 68%, therefore ES-89
For one plant of bacterial strain to having high inhibition to act on to plant pathogenic fungi.
Embodiment 5：
The impact of inhibitory action and mycelial structure of the ES-89 strain fermentations product to Rhizoma Coptidis leaf spot fungi mycelial growth
A, ES-89 strain 1ml are inoculated in 100ml NB culture fluid activation 24h, the ES-89 strain 1ml of activation are connect
Plant in the 250ml triangular flasks containing 100ml NB culture fluid, 28 DEG C, 160rpm, shaken cultivation 48h.Ferment into which after shaking training
Liquid, fermentation liquid is placed under the conditions of 4 DEG C, and 8000rpm is centrifuged 20min, and biofilter (0.22um) filters high fermentation liquid, obtains
Arrive aseptic fermented supernatant fluid.
B, by fermented supernatant fluid by 1%, 5%, 10%, 15%, 20%, 25% addition PDA in be down flat plate, on each flat board
The Rhizoma Coptidis leaf spot fungi mycelia block of a diameter of 6mm is connect, 3 repetitions of each process, 3 parallel tests, 25 DEG C of cultures are surveyed after 5d
The colony diameter of amount Rhizoma Coptidis leaf spot fungi simultaneously records related data, while picking mycelia basis of microscopic observation Rhizoma Coptidis leaf spot fungi bacterium
The metamorphosis of silk.
C, ES-89 tunning shows (Fig. 5) that to the inhibition test of Rhizoma Coptidis leaf spot fungi mycelial growth ES-89 bacterial strains are not
Rhizoma Coptidis leaf spot fungi mycelial growth can be suppressed with concentration tunning, and gradually strengthened with the increase suppression ratio of concentration.
On the PDA plate containing 1%, 5%, 10%, 15%, 20%, 25% (V/V) fermented supernatant fluid, compared with the control, which suppresses hundred
Rate is divided to be respectively 8%, 30%, 52%, 70%, 88%, 95%.There is significant difference (P between each process<0.05).
When D, 5d, examine under a microscope that pathogen mycelial growth in control PDA culture medium is vigorous, mycelia coloring is
Even, finer and close bacterium colony is formed, edge is more neat, and branch is less；Rhizoma Coptidis leaf spot fungi in the culture medium of tunning containing ES-89
Mycelia cluster is given birth to, and branch is more, mycelia deformity, ultimate swelling, mycelia cells in vivo matter skewness (Fig. 6).
Embodiment 6：ES-89 bacterial strains are in vitro prevention effect in Rhizoma Coptidis leaf spot room
A, the healthy Rhizoma Coptidis blade for selecting insect infection in the same size, disease-free, tap water rinse well after through 70% (V/V) wine
Smart surface sterilization 30s, aseptic water washing three times, dries.
B, ES-89 strain 1ml are inoculated in 100mlNB activation 24h, the ES-89 strain 1ml of activation are inoculated into and are equipped with
In the 250ml triangular flasks of 100ml NB, 28 DEG C, 160rpm, shaken cultivation 48h.Its tunning is obtained after shaking training, adds 1%
(V/V) polysorbas20.
C, take surface by the Rhizoma Coptidis blade of the health of alcohol disinfecting, be placed on be covered with moistening absorbent paper porcelain dish (35 ×
In 50cm), by ES-89 tunnings even spraying in Rhizoma Coptidis blade, dry naturally, sprayed as control using sterilized water, each
Process sets 10 blades, 3 repetitions.Then by the Rhizoma Coptidis of 6mm Rhizoma Coptidis Leaf blotch pathogeny bacterium inoculated by hypha block to each treatment group
On blade, moisturizing is sealed using preservative film, be placed in 25 DEG C of constant temperature illumination box cultures, measure Lesion size after 48h.Test knot
Fruit sees Fig. 8 and Biao 2.
2 in vitro ES-89 bacterial strain fermentation liquors of table are to Rhizoma Coptidis leaf spot suppression ratio (5d, 25 DEG C)
In excised leaf test, matched group is inoculated with Rhizoma Coptidis leaf spot fungi mycelia block, starts fleck occur, after 48h after 24h
Just black scab is formed, site of pathological change starts to decay afterwards, in wilting, Lesion size reaches 1.5-2.1cm's for part of not falling ill
Length.ES-89 fermentation liquor treatment group scabs mean size is 0.32cm, and suppression ratio reaches 80.2%.ES-89 strain fermentations are described
Product is acted on very high inhibition to the extension of Rhizoma Coptidis leaf spot.
Embodiment 7：ES-89 bacterial strains are to the potted plant efficiency test of Rhizoma Coptidis leaf spot
Modeling from Enshi State of Hubei Province Taishan mausoleum Rhizoma Coptidis GAP planting bases transplant health Rhizoma Coptidis plant to (30cm × 20cm)
In feed basin.The aseptic fermented supernatant fluid preparation method of ES-89 is with embodiment 3；Mechanical damage mycelia method is adopted in PDA culture medium
Carry out induction and produce spore, after spore is produced, add 10ml sterilized water per ware, concentration is made into for 1 × 106The conidial suspension of individual/ml
(containing 1% tween).Per plant of Rhizoma Coptidis leaf spot fungi conidial suspension for uniformly spraying 20ml, sprinkling equivalent after natural air drying
ES-89 fermentation liquids, 10% (V/V) Difenoconazole 1500 times of liquid, sterilized water.Repeat to test 3 times, 25 DEG C of cultures.Test process
Observe the incidence of Rhizoma Coptidis, Investigate incidence and disease index after 5d respectively, calculate prevention effect.
Rhizoma Coptidis leaf spot grade scale：
0 grade：Blade disease-free spot
1 grade：Lesion area accounts for the 25% of integrated plate blade
2 grades：Lesion area accounts for more than the 25% of integrated plate blade, and less than 50%
3 grades：Lesion area accounts for integrated plate blade more than 50%, and less than 75%
4 grades：Lesion area accounts for integrated plate blade more than 75%
Disease index=(the sick numbers of sheets at different levels × series/total number of sheets of falling ill accordingly × highest morbidity series) × 100
Prevention effect (%)=(the average state of an illness of the average disease index/matched group of the average disease index-treatment group of matched group
Index) × 100%
Table 3ES-89 bacterial strains are to the potted plant prevention effect of Rhizoma Coptidis leaf spot (5d, 25 DEG C)
Results from pot experiment test such as Fig. 9, table 3, after inoculation Rhizoma Coptidis leaf spot fungi 24h, occur on matched group Rhizoma Coptidis blade
Fleck, then fleck slowly expand to black scab, after inoculation 5d, black scab slowly becomes regular or irregularly dark brown
The big scab of color, vacuum side of blade assume yellowish-brown.Control chemical agent 10% Difenoconazole process after Rhizoma Coptidis blade sickness rate very
Low, relative to matched group, the Rhizoma Coptidis blade state of an illness of ES-89 fermentation liquor treatments is lighter, only has a small amount of scab and occur after 48h, and disease
Speckle is less, the slow sickness rate of expansion rate is only 12.5%, and average disease index is 3.1, and prevention effect reaches 88.5%.Explanation
ES-89 fermentation liquids have preferable prevention effect to Rhizoma Coptidis leaf spot.
SEQUENCE LISTING
<110>Hua Zhong Agriculture University
<120>A kind of bulkholderia cepasea bacterial strain ES-89 of preventing and treating fungal diseases of plants and cultural method and application
<130>A kind of bulkholderia cepasea bacterial strain ES-89 of preventing and treating fungal diseases of plants and cultural method and application
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1336
<212> DNA
<213>Bulkholderia cepasea ES-89
<400> 1
atacatcgga acatgtcctg tagtggggga tagcccggcg aaagccggat taataccgca 60
tacgatctac ggatgaaagc gggggacctt cgggcctcgc gctatagggt tggccgatgg 120
ctgattagct agttggtggg gtaaaggcct accaaggcga cgatcagtag ctggtctgag 180
aggacgacca gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg 240
gggaattttg gacaatgggc gaaagcctga tccagcaatg ccgcgtgtgt gaagaaggcc 300
ttcgggttgta aagcacttt tgtccggaaa gaaatccttg gttctaatat aaccggggga 360
tgacggtacc ggaagaataa gcaccggcta actacgtgcc agcagccgcg gtaatacgta 420
gggtgcgagc gttaatcgga attactgggc gtaaagcgtg cgcaggcggt ttgctaagac 480
cgatgtgaaa tccccgggct caacctggga actgcattgg tgactggcag gctagagtat 540
ggcagagggg ggtagaattc cacgtgtagc agtgaaatgc gtagagatgt ggaggaatac 600
cgatggcgaa ggcagccccc tgggccaata ctgacgctca tgcacgaaag cgtggggagc 660
aaacaggatt agataccctg gtagtccacg ccctaaacga tgtcaactag ttgttgggga 720
ttcatttcct tagtaacgta gctaacgcgt gaagttgacc gcctggggag tacggtcgca 780
agattaaaac tcaaaggaat tgacggggac ccgcacaagc ggtggatgat gtggattaat 840
tcgatgcaac gcgaaaaacc ttacctaccc ttgacatggt cggaatcctg ctgagaggcg 900
ggagtgctcg aaagagaacc gatacacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt 960
gagatgttgg gttaagtccc gcaacgagcg caacccttgt ccttagttgc tacgcaagag 1020
cactctaagg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt caagtcctca 1080
tggcccttat gggtagggct tcacacgtca tacaatggtc ggaacagagg gttgccaacc 1140
cgcgaggggg agctaatccc agaaaaccga tcgtagtccg gattgcactc tgcaactcga 1200
gtgcatgaag ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1260
gggtcttgta cacaccgccc gtcacaccat gggagtgggt tttaccagaa gtggctagtc 1320
taaccgcaag gaggac 1336
Claims (6)
1. a kind of biocontrol bacteria bulkholderia cepasea ES-89, it is characterised in that：Bulkholderia cepasea Burkholderia
Sp, CCTCC, CCTCC NO：M 2016665.
2. a kind of biocontrol bacteria bulkholderia cepasea ES-89 according to claim 1, it is characterised in that：Described primary
Ke Huoerde Salmonella ES-89 on nutrient agar bacterium colony be milky, neat in edge, subcircular, smooth surface swell,
Opaque, in oily, cell is oval to circle, and 0.5-0.6 × 1-2 μm, Gram’s staining reaction is redness, is gram-negative
Property antibacterial.
3. a kind of bulkholderia cepasea bacterial strain ES-89 according to claim 1, it is characterised in that：Described Bai Kehuo
Your moral Salmonella contains its sequence for the nucleotide sequence shown in SEQ ID NO.2.
4. the cultural method of the bulkholderia cepasea bacterial strain ES-89 of a kind of preventing and treating fungal diseases of plants described in claim 1,
Its step is as follows：
A, ES-89 strain 1ml are inoculated in 100ml nutrient broth culture fluid activation 22-26h；
B, the ES-89 strain 1ml of activation in step (A) are inoculated into the 250ml triangular flasks containing 100ml nutrient broth culture fluid
In, 26-30 DEG C, 160rpm, shaken cultivation 46-50h；
C, by shake in step (B) training after obtain fermentation liquid be placed in 4 DEG C under the conditions of, 8000rpm centrifugation 18-22min, antibacterial mistake
Filter filters high fermentation liquid, obtains aseptic fermented supernatant fluid；
D, thalline is washed 2-4 time with sterilized water, make its thalline suspension, fermentation liquid, fermented supernatant fluid, thalline suspension are put
Preserve under the conditions of 4 DEG C, standby；
Described nutrient broth culture fluid component and formula are：Beef extract 5g, peptone 10g, sucrose 10g, supplement distilled water
To 1000ml, pH to 7.2-7.4 is adjusted, in 121 DEG C of high pressure steam sterilization 30min；
Described nutrient agar component and formula are：Beef extract 5g, peptone 10g, sucrose 10g, add agar 15-
20g, supplements distilled water to 1000ml, adjusts pH to 7.2-7.4, in 121 DEG C of high pressure steam sterilization 28-32min；
Described potato dextrose agar component and formula are：Rhizoma Solani tuber osi 200g, glucose 20g, add agar 15-
20g, supplements distilled water to 1000ml, and natural pH, in 121 DEG C of high pressure steam sterilization 30min.
5. a kind of bulkholderia cepasea bacterial strain ES-89 of the preventing and treating fungal diseases of plants described in claim 1 is preparing treatment
Or the application in prevention plant pathogenic fungi medicine；Described plant pathogenic fungi is Rhizoma Coptidis leaf spot fungi, grape ulcer disease
Bacterium, tobacco brown spot pathogen, Pyricularia oryzae, Flos Ilicis Asprellae anthrax bacteria.
6. a kind of bulkholderia cepasea bacterial strain ES-89 of the preventing and treating fungal diseases of plants described in claim 1 is preparing treatment
Or the application in the medicine of prevention Rhizoma Coptidis leaf spot.
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| CN107058125A (en) * | 2017-03-21 | 2017-08-18 | 三峡大学 | One plant of aspergillus niger and its application with rice blast antagonism |
| CN107267424A (en) * | 2017-08-02 | 2017-10-20 | 上海乾界生物科技有限公司 | A kind of process for preparing microbial insecticide |
| CN108384734A (en) * | 2018-02-22 | 2018-08-10 | 广西壮族自治区农业科学院微生物研究所 | A kind of biocontrol agent and preparation method thereof of prevention tobacco rhizome class disease |
| CN109234202A (en) * | 2018-10-29 | 2019-01-18 | 华侨大学 | A kind of fermentation culture method of banana blight Antagonistic Fungi bulkholderia cepasea |
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Cited By (6)
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|---|---|---|---|---|
| CN107058125A (en) * | 2017-03-21 | 2017-08-18 | 三峡大学 | One plant of aspergillus niger and its application with rice blast antagonism |
| CN107058125B (en) * | 2017-03-21 | 2019-08-13 | 三峡大学 | One plant of aspergillus niger and its application with rice blast antagonism |
| CN107267424A (en) * | 2017-08-02 | 2017-10-20 | 上海乾界生物科技有限公司 | A kind of process for preparing microbial insecticide |
| CN108384734A (en) * | 2018-02-22 | 2018-08-10 | 广西壮族自治区农业科学院微生物研究所 | A kind of biocontrol agent and preparation method thereof of prevention tobacco rhizome class disease |
| CN108384734B (en) * | 2018-02-22 | 2021-04-30 | 广西壮族自治区农业科学院微生物研究所 | Biocontrol microbial inoculum for preventing and treating tobacco root and stem diseases and preparation method thereof |
| CN109234202A (en) * | 2018-10-29 | 2019-01-18 | 华侨大学 | A kind of fermentation culture method of banana blight Antagonistic Fungi bulkholderia cepasea |
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