CN108220180A - A kind of bacillus amyloliquefaciens Substance extracting method and its application as microbial inoculum in rice blast biological and ecological methods to prevent plant disease, pests, and erosion - Google Patents
A kind of bacillus amyloliquefaciens Substance extracting method and its application as microbial inoculum in rice blast biological and ecological methods to prevent plant disease, pests, and erosion Download PDFInfo
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- CN108220180A CN108220180A CN201611151513.8A CN201611151513A CN108220180A CN 108220180 A CN108220180 A CN 108220180A CN 201611151513 A CN201611151513 A CN 201611151513A CN 108220180 A CN108220180 A CN 108220180A
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Abstract
A kind of application the present invention provides bacillus amyloliquefaciens Substance extracting method and its as microbial inoculum in rice blast biological and ecological methods to prevent plant disease, pests, and erosion.A kind of preferred bacillus amyloliquefaciens are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC No.11488.Application provided by the invention is to prevent rice blast using the Substance of the bacillus amyloliquefaciens generation.The bacillus amyloliquefaciens Substance extraction flow being capable of quickly and easily effective component extracting, and with higher inhibition rice blast fungus activity.The Substance exploitation prevents rice blast after microbial inoculum, to can be used in, and provides a kind of rice blast biological and ecological methods to prevent plant disease, pests, and erosion method of efficient stable, can preferably meet the requirement that rice blast is prevented in agricultural production.
Description
Technical field
The present invention relates to a kind of bacillus amyloliquefaciens Substance extracting methods more particularly to one kind to contain Xie Dian
Application of the aqua of afnyloliquefaciens Substance in rice blast prevention.
Background technology
Rice blast is the important disease of rice, and pathogen can infect rice leaf, stalk, fringe portion, be divided into seedling pest, leaf pest,
Save pest, panicle blast, grain pest.Rice blast is happened at rice seedling seedling stage and tillering stage, rice leaf can be caused largely withered, rice
Plant atrophy is not eared or ears short and small.Rice blast is fallen ill at booting heading stage, causes section pest, panicle blast, can cause to generate big
It measures dead ears or half dead ears, production loss is very big.Rice blast has different degrees of generation, popular time in China south and north rice region
General underproduction 10-20%, the serious underproduction reach 40-50%.
Rice blast prevention at present is based on chemical prevention, such as thiophanate methyl wettable powder, tricyclazole wettable powder
Deng.However, the problems such as due to the drug resistance of Pyricularia oryzae and serious chemical prevention environmental pollution, carry out the biology of rice blast
Study on prevention is of great significance.
At present, having screened some has Pyricularia oryzae the microorganism of Biocontrol Effect.
Chinese patent application CN105420156A discloses one plant of solution starch gemma bar with phosphorus decomposing disease prevention growth-promoting effect
Bacterium (Bacillus amyloliquefaciens), is preserved in China typical culture collection center.The bacterium can effectively prevent white
It is powder disease, tobacco black shank, gray mold, rice blast, cucumber fusarium axysporum, early blight of tomato, pimento anthracnose, watermelon sclerotiniose, small
Wheat head blight, watermelon blight, P. capsici etc., broad-spectrum antiseptic and stable and durable effect.
Chinese patent application CN104974965A discloses a bacillus subtilis JN005 and its in prevention rice rice blast
Application in disease.China typical culture collection center is preserved in, preserving number is CCTCC No：M2015228.Bacterial strain JN005 lives
Body bacterium and its metabolite have rice blast preferable preventive effect, and protection and therapeutic effect have both, can be as living microbial inoculum
It is developed with the formulation of metabolite.
Although having screened some has Pyricularia oryzae the microorganism of Biocontrol Effect, also far from enough.On the one hand,
The diversified use of antagonistic microbe is conducive to prevent germ from generating resistance in biological and ecological methods to prevent plant disease, pests, and erosion；On the other hand, it is existing to inhibit rice
The antagonism of the microorganism of seasonal febrile diseases bacterium is preferable not enough, it is impossible to preferably meet and wanting for rice blast is prevented in agricultural production
It asks.Biocontrol microorganisms living microbial inoculum production, transport, preserve and application in there are limitation, due to thalline environmental condition is required compared with
Height, and it is also selective to plant, it is easily influenced in Field information by environment, so as to reduce control effect.From biocontrol microorganisms
Middle extraction active material, is applied to as medicament in production, reduces the link that thalline is preserved and is colonized on plant, improves
Medicament utilization rate.
Invention content
The object of the present invention is to provide a kind of extracting methods of bacillus amyloliquefaciens bacteriostatic activity product.
It is a further object to provide application of the above-mentioned bacteriostatic activity product in rice blast biological and ecological methods to prevent plant disease, pests, and erosion, significantly to carry
Rise rice blast control effect.
It is also another object of the present invention to provide a kind of biological prevention and control agents, and preparation process is simple, and can be obviously improved rice
Seasonal febrile diseases biocontrol effect.
It is common that bacillus amyloliquefaciens according to the present invention are preserved in China Committee for Culture Collection of Microorganisms
Microorganism center, preserving number are CGMCC No.11488.
According to the application provided by the invention, bacillus amyloliquefaciens zymotic fluid contains Substance, Neng Gouyou
Effect is suppressed to rice blast fungus growth.
According to the application provided by the invention, will be precipitated containing the bacillus amyloliquefaciens zymotic fluid through HCL, methanol
It extracts, stays and be precipitated as containing high concentration Substance after rotary evaporation, can effectively be suppressed to rice blast fungus growth.
Application according to the present invention, sediment and ultra-pure water after the bacillus amyloliquefaciens zymotic fluid methanol extracting
It is configured to a concentration of 5% aqua.
Application according to the present invention, it is preferable that be that will contain the water by the method that aqua is imposed on rice plant
It sprays after 200 times of dilution agent.
Application according to the present invention, it is preferable that the amount of spraying of the hydrojet is 0.2~0.5 liter/mu.
The bacillus amyloliquefaciens and its zymotic fluid of the present invention have stronger inhibiting effect to Pyricularia oryzae.Profit of the invention
Contain Substance with bacillus amyloliquefaciens zymotic fluid methanol extracting sediment, can effectively inhibit Pyricularia oryzae.This
Invention is configured to 5% aqua using bacillus amyloliquefaciens zymotic fluid methanol extracting sediment, is administered on rice and prevents rice
Seasonal febrile diseases.The Substance that the bacillus amyloliquefaciens generate, which is utilized, has Pyricularia oryzae the spy of strong antagonism
Point can preferably meet the requirement that rice blast is prevented in agricultural production.
Description of the drawings
Fig. 1 is Pyricularia oryzae and bacillus amyloliquefaciens opposite culture result；
Fig. 2 is for examining bacillus amyloliquefaciens zymotic fluid to control and processing item in the inhibiting effect experiment of rice blast fungus
Pyricularia oryzae colonial morphology under part.
Fig. 3 is that the inhibiting effect of rice blast fungus is tested for bacillus amyloliquefaciens zymotic fluid methanol to be examined to extract sediment
Pyricularia oryzae colonial morphology under middle control and treatment conditions.
Fig. 4 is that rice blast fungus mycelia extracts the form under the effect of sediment aqueous solution in bacillus amyloliquefaciens zymotic fluid methanol
Variation.
Specific embodiment
Below in conjunction with the accompanying drawings and specific embodiment the present invention is further illustrated, but protection scope of the present invention is simultaneously
It is without being limited thereto.
<Bacillus amyloliquefaciens>
Bacillus amyloliquefaciens provided by the invention have been preserved in budapest treaty microorganism International Depository Authority：China
Microbiological Culture Collection administration committee common micro-organisms center (CGMCC).Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica, postcode：100101.Preservation date on October 13rd, 2015, preserving number is CGMCC
No.11488。
<Bacillus amyloliquefaciens and its zymotic fluid are to rice blast fungus inhibiting effect>
Using opposite culture method Preliminary detection bacillus amyloliquefaciens to the inhibiting effect of rice blast fungus.Using filter paper enzyme into
Inhibitory activity of the bacillus amyloliquefaciens zymotic fluid to rice blast fungus is determined in one pacing, inhibits rice blast fungus to contain in clear and definite zymotic fluid
Active material.
<Bacillus amyloliquefaciens Substance extracts flow>
Bacillus amyloliquefaciens Substance is mostly cyclic lipopeptide.Therefore starch gemma bar will tentatively be solved using HCL
Albumen and peptide matters species precipitate in fermented liquid.Then cyclic lipopeptide substance is extracted using the highly polar feature of methanol,
After rotary evaporation, sediment is stayed to contain high concentration Substance.
<Bacillus amyloliquefaciens zymotic fluid methanol extracts sediment to rice blast fungus inhibitory activity>
Suppression of the bacillus amyloliquefaciens zymotic fluid methanol extracting sediment to rice blast fungus is further measured using filter paper enzyme
System activity, to contain the active material for inhibiting rice blast fungus in clear and definite sediment.
<Bacillus amyloliquefaciens zymotic fluid methanol extracts application of the sediment in rice blast biological and ecological methods to prevent plant disease, pests, and erosion>
Bacillus amyloliquefaciens zymotic fluid methanol extracting sediment will be contained and be configured to 5% aqua, after 200 times of dilution
It imposes on rice leaf, prevents rice blast.It can be any period by the period imposed on containing the aqua on rice leaf；It is excellent
It is selected as rice blast leaf pest and early period occurs for panicle blast, be the critical period for preventing rice blast at this time.
Spraying method commonly employed in the art is may be used into the method imposed on rice plant containing the aqua agent,
That is, it will be sprayed onto on rice plant containing the aqua hydrojet using spraying apparatus.The amount of spraying of the hydrojet can be 0.2~
0.5 liter/mu, is preferred with repeatedly spraying.
Bacillus amyloliquefaciens Substance in the aqua of the hydrojet is mostly cyclic lipopeptide, belongs to antibiotics
Fungicide.Therefore it should be stored at shady and cool drying, and pay attention to anti-corrosion, mould proof, solar heat protection.It deposits in shady and cool, dry warehouse, and
Pay attention to mould proof, solar heat protection, antifreeze.
Below by embodiment, the present invention will be further described.
Embodiment 1
The bacillus amyloliquefaciens of the present invention are to rice blast fungus inhibitory activity Preliminary Determination：
The bacteria cake for taking diameter 0.5cm is beaten in the rice blast fungus colony edge of culture 4d with card punch, is transferred on PDA plate,
5 μ L of bacillus amyloliquefaciens bacterium solution are met apart from bacterium colony 2.5cm points, are placed in 25 DEG C of cultures.Three repetitions are set.After cultivating 5d, see
Bacterium colony upgrowth situation is examined, and measures inhibition zone size.
LB culture medium 100mL, the 50 μ L of bacillus amyloliquefaciens bacterium solution of access culture 12h are added in 250mL triangular flasks.
On 30 DEG C, rotating speed 150rmp/min shaking tables, bacillus amyloliquefaciens shake culture 38h, then zymotic fluid centrifuged through 10000g
5min, removes thalline, and the filtering of 0.22 μ L biofilters of supernatant via hole diameter is spare as ferment filtrate.By rice blast fungus bacteria cake
It is transferred on PDA plate, 4 0.5cm diameter aseptic filter paper pieces is taken to be placed in around rice blast bacteria cake, apart from bacteria cake 2cm.In filter paper
10 μ L ferment filtrates of upper dropwise addition, culture dish are placed in 25 DEG C of cultures.The LB culture solutions of 10 μ L are added dropwise in control treatment, filter paper.Setting 3
A repetition and blank control.After 5d, processing bacterium colony size and inhibition zone width are measured.
With reference to Fig. 1, rice blast fungus mutually closes on side with bacillus amyloliquefaciens, and bacterium colony growth is suppressed, it is difficult to be extended.Rice
Pest bacterium is apart from the distant side of bacillus amyloliquefaciens, mycelia normal growth.This shows that bacillus amyloliquefaciens can be effective
Inhibit rice blast fungus growth.With reference to Fig. 2, under LB culture solution control treatments, rice blast fungus normal growth, under fermentation liquor treatment, rice blast fungus
Bacterium colony growth is suppressed, it is difficult to be extended, bacterium colony is in quadrangle.This shows to contain bateriostatics in bacillus amyloliquefaciens zymotic fluid
Matter effectively can inhibit rice blast fungus to grow.
Embodiment 2
Identification of the bacillus amyloliquefaciens zymotic fluid methanol extracting sediment of the present invention to Pyricularia oryzae inhibiting effect：
HCL is added in the bacillus amyloliquefaciens ferment filtrate obtained in embodiment 1, pH is adjusted to 2, is then dispensed into
In 50mL centrifuge tubes, 4 DEG C of refrigerator overnight precipitations.Then 4 DEG C, 10000g centrifugation 5min abandon supernatant, collect precipitation, add in
3.5mL methanol will precipitate gravity treatment in vortex instrument, extract 8h.Then 4 DEG C, 10000g centrifugation 5min take supernatant.Same method
It is extracted twice, merges supernatant.It is distilled in Rotary Evaporators, removes methanol, precipitated, precipitated with ultrapure water dissolution, it is spare.
The bacteria cake for taking diameter 0.5cm is beaten in the rice blast fungus colony edge of culture 4d with card punch, is transferred on PDA plate,
4 0.5cm diameter aseptic filter paper pieces is taken to be placed in around rice blast bacteria cake, apart from bacteria cake 2cm.10 μ L extractions are added dropwise on filter paper
Object, culture dish are placed in 25 DEG C of cultures.The LB culture solutions of 10 μ L are added dropwise in control treatment, filter paper.3 repetitions and blank pair are set
According to.After 5d, processing bacterium colony size and inhibition zone width are measured.It can be seen that according to antibacterial bandwidth size and bacterium colony growing state
To the inhibition strength of rice blast.
Referring to Fig. 3, bacillus amyloliquefaciens zymotic fluid methanol of the invention extracting sediment significantly inhibits rice blast fungus bacterium colony
Growth, and bacterium colony normal growth is compareed, show that its growth inhibition efficiency to rice blast fungus is higher.Referring to Fig. 4, rice blast fungus mycelia exists
Under processing, mycelia enlarges into spherical shape, it is difficult to and further growth illustrates that Substance destroys the cell wall of mycelia, so as to
Inhibit mycelia growth.
Embodiment 3
The bacillus amyloliquefaciens zymotic fluid methanol extracting sediment of the present invention identifies rice blast control efficacy：
Bacillus amyloliquefaciens methanol extracting sediment is configured to 5% aqua.Rice varieties use Nipponbare, to rice
Seasonal febrile diseases height sense.Rice paddy seed with clear water is dipped at room temperature and is showed money or valuables one carries unintentionally, is seeded into after vernalization 10h equipped with turf and leech at 37 DEG C
In the nutritive cube of stone (2: 1).It is used when rice length to the 5th leaf will be fully deployed for inoculation.
Pyricularia oryzae is seeded in PDA culture medium, 7d is cultivated under the conditions of 25 DEG C, mitogenetic armful is gently washed down with sterile water
Son, centrifugation, abandons supernatant, and conidium is suspended with aqueous solution, and blood counting chamber counts, and conidial suspension concentration is adjusted to 106/
ML spores.
Aqua dilution is sprayed to rice leaf for 200 times, is advisable with dripping.After for 24 hours, then by spray-on process by rice blast fungus point
Raw spore inoculating is to rice leaf, and a concentration of 106A/mL.It compares as sterile water, is then inoculated with same rice blast fungus conidium
Suspension.Environment temperature is 28~30 DEG C, relative humidity 80%.Rice leaf incidence is inspected periodically, counts disease index,
Calculate preventive effect.Leaf pest grade scale：It is 0 grade, disease-free.1 grade, leaf spot lesion few (be less than 5) and it is small (being less than 1cm).2 grades, blade
Scab small and more (being more than 5) (is more than 1cm) greatly and lacks.3 grades, leaf spot lesion is big and more.4 grades, full leaf disease is withered.Utilize this hair
The rice leaf rice blast incidence disease index of the aqua processing of bright embodiment 3 is 10, and it is 36 to compare lower disease index, real
The average preventive effect for applying the suspension of example 3 is 72.2%.
Embodiment 4
The bacillus amyloliquefaciens zymotic fluid methanol extracting sediment of the present invention identifies rice blast field efficacy：
Spray-on process after aqua dilutes 200 times, control spray clear water.Experiment sets 10 cells, plot area 5.0m altogether2, processing 5
A cell compares 5 cells.Rice transplanting specification 25cm × 10cm, dose：Urea 100kg/hm2, Diammonium phosphate (DAP) 80kg/hm2, sulphur
Sour potassium 45kg/hm2.10d carries out 2 even sprayings after rice blast early stage and the 1st spray.The amount of spraying can
Think 0.2~0.5 liter/mu.Leaf pest and panicle blast incidence are investigated after 14d.Per 5 points of samplings of cell, every takes 20 plants, every plant
Investigate blade and the incidence of fringe neck.According to grade scale, disease index and field efficacy are calculated.Panicle blast and section pest classification
Standard：It is 0 grade, disease-free.1 grade, less than 5% loss (indivedual sprig stalk morbidities) per fringe.2 grades, per fringe, (1/3 is left for loss 20% or so
Right sprig stalk morbidity).3 grades, loss 50% or so per fringe (fringe neck or main shaft morbidity, grain half are flat).4 grades, 70% is lost per fringe
Left and right (fringe neck is fallen ill, big portion's shrivelled kernel).5 grades, loss 90% or so per fringe (fringe neck is fallen ill, and causes dead ears).
After the aqua of the embodiment of the present invention 4 sprays field, leaf pest disease index is 11, and it is 32 to compare lower disease index,
Average preventive effect is 65.6%.Panicle blast disease index is 16, and compares lower disease index 42, and average preventive effect is 61.9%.
Present invention is not limited to the embodiments described above, in the case of without departing substantially from the substantive content of the present invention, this field skill
Any deformation, improvement, the replacement that art personnel are contemplated that each fall within the scope of the present invention.
Claims (7)
1. the bacillus amyloliquefaciens are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation
Number be CGMCC No.11488.
2. application according to claim 1, which is characterized in that the bacillus amyloliquefaciens and its zymotic fluid are to rice blast fungus
With stronger inhibiting effect.
3. application according to claim 2, which is characterized in that bacillus amyloliquefaciens zymotic fluid is through hydrochloric acid precipitation, methanol
After extracting, rotary evaporation is stayed in precipitation containing the higher Substance of concentration.
4. application according to claim 3, which is characterized in that the methanol that the aqua is 5% extracts sediment aqueous solution.
5. application according to claim 4, which is characterized in that the method that will be imposed on containing the aqua on rice plant
For that will contain hydrojet after the aqua dilutes 200 times and spray.
6. application according to claim 5, which is characterized in that the amount of spraying of the hydrojet is 0.2~0.5 liter/mu.
7. a kind of biological prevention and control agent, which is characterized in that for the aqua in application described in claim 5 or 6.
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| CN201611151513.8A CN108220180A (en) | 2016-12-14 | 2016-12-14 | A kind of bacillus amyloliquefaciens Substance extracting method and its application as microbial inoculum in rice blast biological and ecological methods to prevent plant disease, pests, and erosion |
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| CN201611151513.8A CN108220180A (en) | 2016-12-14 | 2016-12-14 | A kind of bacillus amyloliquefaciens Substance extracting method and its application as microbial inoculum in rice blast biological and ecological methods to prevent plant disease, pests, and erosion |
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| CN104630086A (en) * | 2014-11-20 | 2015-05-20 | 中国农业大学 | Bacillus amyloliquefaciens RL263 capable of preventing and controlling rice blast |
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| CN105420156A (en) * | 2015-12-19 | 2016-03-23 | 佛山市艳晖生物科技有限公司 | Bacillus amyloliquefaciens with phosphate solubilizing, disease preventing and growth promoting functions and application thereof |
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| KR20130005322A (en) * | 2011-07-06 | 2013-01-16 | 재단법인 전라남도생물산업진흥재단 | Bacillus amyloliquefaciens lm11 strain, composition for control plant disease and control method of plant disease with same |
| CN102604864A (en) * | 2012-03-09 | 2012-07-25 | 陕西加伦多作物科学有限公司 | Microbial bactericide and application thereof |
| CN104630086A (en) * | 2014-11-20 | 2015-05-20 | 中国农业大学 | Bacillus amyloliquefaciens RL263 capable of preventing and controlling rice blast |
| CN105349457A (en) * | 2015-11-17 | 2016-02-24 | 中国农业科学院植物保护研究所 | Bacillus amyloliquefaciens and application thereof to biocontrol on northern corn leaf blight |
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Application publication date: 20180629 |