CN108048357A - A kind of bacillus subtilis and its cultural method - Google Patents
A kind of bacillus subtilis and its cultural method Download PDFInfo
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- CN108048357A CN108048357A CN201711432174.5A CN201711432174A CN108048357A CN 108048357 A CN108048357 A CN 108048357A CN 201711432174 A CN201711432174 A CN 201711432174A CN 108048357 A CN108048357 A CN 108048357A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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 104 bacterial strains of bacillus subtilis (Bacillus subtilis) KN, which is deposited in China typical culture collection center, and deposit number is CCTCC NO:M2017697.By orienting target sieving, the high specificity of disease-resistant kind of obtained bacillus subtilis, there is significant biocontrol effect to rice blast, leaf seasonal febrile diseases, rice sheath blight disease, Cabbage Leaf Spot, but there is no preventive effect to diseases such as graw mold of tomato, watermelon blight, apple anthracnose, root rot, leafs muld of tomato, while the fertility of the bacterial strain is strong and without hemolytic.
Description
Technical field
The present invention relates to biological pesticide and technical field of fertilizers, particularly bacillus subtilis (Bacillus
Subtilis screening and application).
Background technology
The main means of prevention corps diseases are plantation disease-resistant variety, using chemical pesticide and using biological agriculture at present
Medicine.And main chemical pesticide such as tricyclazole, rice blast missible oil, carbendazim, thiophanate methyl, Bravo etc. easily generate germ
The resistance to the action of a drug makes preventive effect constantly decline, and improper use can also bring very important serious harm to ecological environment, human health,
Therefore biological control is increasingly becoming the main means for crop disease.
Bacillus subtilis is one kind of bacillus, due to Bacillus subtillis have breeding is fast, colonization ability is strong,
To person poultry harmless, environment compatibility is good, the advantages that can inhibiting plurality of plant diseases, be not easy that crops is made to develop immunity to drugs, is
One of biocontrol microorganisms being most widely used at present.But most of bacillus subtilises are because have hemolytic at present, and cannot
Applied to biological fertilizer field.CN 101121924A disclose a kind of bacillus subtilis can be used for prevention by wheat scab,
The diseases such as crops, vegetables caused by rice blast, corn southern leaf blight, cucumber anthracnose etc..Disclosed in CN 102433282A
Bacillus subtilis Rhizoctonia solani, banana anthracnose, rice blast, wheat scab, banana blight, sugarcane black rot,
Leaf muld of tomato etc. has strong inhibitory action, is wide spectrum Antagonistic Fungi above.
In addition, existing fermentation of bacillus subtilis level is constantly in bottleneck period, without very big breakthrough.Zou Gaoxi etc.
(Zhejiang Agriculture journal, 2017,29 (5):799-805) by response surface optimization, spore production just reaches 16,400,000,000 CFU/ml.Merchant
(Journal of Shandong agri.Univ (natural science edition), 2013,44 (1) such as an ancient unit of weight brightness:35-39) Biocontrol Bacillus subtilis B579 is mended
Material batch process is optimized, and cultivates to 40h spore concentrations and also just reaches 28,000,000,000 CFU/ml.
The content of the invention
It is an object of the invention to be directed to shortcoming existing in the prior art, provide a kind of disease resistance specificity it is good, breeding energy
Power is strong and bacillus subtilis strain without hemolytic, and the present invention also provides the cultural methods of the bacterial strain.
The purpose of the present invention is what is be achieved through the following technical solutions：
By orienting target sieving method, using rice blast as pathogeny target, from the rhizosphere soil of rice Experimental Base
In isolated plant height effect controlling disease bacterium, it is identified to belong to typical bacillus subtilis (Bacillus
Subtilis), applicant is named as bacillus subtilis KN-104.The bacterial strain is in place in preservation on November 17 in 2017
In the China typical culture collection center of Wuhan City, Hubei Province Wuhan University, deposit number is CCTCC NO:M2017697.
The experimental results showed that bacillus subtilis KN-104 is to rice blast, Rice Leaf seasonal febrile diseases, rice sheath blight disease, white
Dish black spot have stronger rejection ability, but to graw mold of tomato, watermelon blight, apple anthracnose, root rot, kind
The preventive effect of other all crop diseases such as eggplant leaf mold is very low or without preventive effect, so as to prove the bacillus subtilis of the invention screened
Bacterium KN-104 has stronger specificity in disease prevention species, can more targeted prevention crop disease.
Bacterial strain provided by the present invention also has the advantages that fertility is stronger compared with existing bacillus subtilis,
So as to obtain higher brood cell's number living in the culture of bacterial strain, production cost is advantageously reduced, improves production efficiency.
Most of microbe can generate hemolysin during growth metabolism, and hemolysin can be completely dissolved red blood cell, from
And people and animals are generated with harm, and bacillus subtilis KN-104 provided by the invention is completely without hemolytic, therefore application is more
Safety.
The cultural method of bacillus subtilis KN-104 bacterial strains provided by the invention, comprises the following steps：
(1) actication of culture：
One ring Bacillus subtillis KN-104 bacterial strains of picking, are inoculated on slant medium, and 60h is cultivated in 30 DEG C,
Slant medium：Peptone 10g/L, dusty yeast 5g/L, sodium chloride 10g/L, agar powder 15g/L, pH value 7.0；
(2) seed culture：
The Bacillus subtillis KN-104 that step (1) activates is inoculated in seed culture medium, in 30 DEG C, the item of 150rpm
12h is cultivated under part,
Seed culture medium：Starch 15g/L, corn pulp 15g/L, yeast extract 3g/L, corn flour 8g/L, peptone 6g/
L, dipotassium hydrogen phosphate 0.5g/L, pH7.2；
(3) fermented and cultured：
Cultured seed liquor is accessed in fermentation medium, 40h is cultivated under conditions of 32 DEG C, 250rpm, obtains withered
Careless bacillus KN-104 zymotic fluids, work brood cell's number of the zymotic fluid reach 35,000,000,000 CFU/ml,
Fermentation medium：Starch 30g/L, corn pulp 30g/L, yeast extract 15g/L, corn flour 10g/L, peptone
25g/L, dregs of beans 15g/L, calcium chloride 0.5g/L, magnesium sulfate 0.8g/L, manganese chloride 0.2g/L, potassium dihydrogen phosphate 0.2g/L,
pH7.5。
The work brood cell's number to be fermented using the above method is significantly higher than existing most similar products.
Description of the drawings
Fig. 1 is the hemolytic result of the test of bacillus subtilis.
Specific embodiment
The present invention is described in more detail below by embodiment.
The screening and identification of 1 bacillus subtilis KN-104 of embodiment
1st, the heat treatment screening of strain
Weigh the triangular flask that soil sample (the crops rhizosphere soil for seriously infecting rice blast disease) 1.0g is encased in sterilizing
In, add in culture solution (starch 5-20g/L, corn pulp 10-20g/L, yeast extract 1-10g/L, corn flour 2- that 90ml sterilizes
15g/L, peptone 5-10g/L, dipotassium hydrogen phosphate 0.5-5g/L, pH7.2-7.5), it is 30 DEG C in temperature, rotating speed 120-
After being cultivated 3 days under conditions of 180rpm, after the water-bath 30min in 80 DEG C of thermostat water baths, fully vibration, zymotic fluid is taken to do concentration
Gradient dilution draws 100ul dilutions and is spread evenly across on NA culture mediums (peptone 10g/L, beef extract powder 3g/L, sodium chloride
5g/L, agar powder 15g/L, pH value 7.2), after 30 DEG C are cultivated 3-5 days, picking single bacterium colony microscopy is picked out with withered grass gemma
The bacterial strain of bacillus morphological feature carries out directed screening experiment in next step；
2nd, target sieving experiment is oriented
It using rice blast as pathogeny target, is screened with tablet face-off method, the rice blast cultivated on PDA plate 7 days is used
Aseptic card punch, which is beaten, takes the pure culture biscuits involvng inoculation of a diameter of 6mm central in new PDA plate, same with sterile toothpick at 30mm in distance
When inoculation in the LB culture solutions (peptone 10g/L, dusty yeast 5g/L, sodium chloride 10g/L) culture 10-12h bacterial strain to be sieved,
Each plating 4 is observed after 3-5 days as a result, for there is the bacterial strain of antagonism, straight with vernier caliper measurement inhibition zone
Footpath.Finally, screening obtains one plant of bacterial strain KN-104 that can effectively inhibit rice blast.
3rd, strain idenfication
(1) bacterial strain 16S rDNA Sequence Identifications
Colony morphology characteristic and physio-biochemical characteristics research are carried out to the bacterial strain that screening obtains in step (2), according to this
16S rDNA sequence of the bacterial strain in Genebank carries out molecular classification, and the 16S rDNA of the bacterial strain measure 1453 effective alkalis altogether
Base, it is bacillus subtilis to identify the bacterial strain.
The bacillus subtilis KN-104 has following feature：
The bacterial strain thalline is rod-shaped, produce gemma, Gram-positive, NA culture mediums (peptone 10g/L, beef extract powder 3g/L,
Sodium chloride 5g/L, agar powder 15g/L, pH value 7.2) on bacterium colony be creamy white or faint yellow, colony edge is irregular, partially flat impermeable
It is bright.
Physiology and biochemistry identification characteristic shows the bacterial strain as aerobic bacteria, and contact enzyme positive, V-P are positive, production amylase；It is unfavorable
With sodium citrate and lactose.
4th, hemolytic is tested
Test procedure is as follows：
1) test principle：Bacterium colony Hemolysis characteristic：1. α haemolysis：Also known as grass green haemolysis, periphery of bacterial colonies culture medium appearance 1~
The grass green colour circle of 2mm, caused by ferrihemoglobin；2. β haemolysis：Also known as complete hemolysis, periphery of bacterial colonies form one completely clearly
Clear transparent zone of hemolysis is that bacteriogenic hemolysin makes caused by red blood cell is completely dissolved；3. γ haemolysis：That is not haemolysis, bacterium colony
The culture medium of surrounding does not change, and red blood cell does not dissolve or defect.
2) subjects：Bacillus subtilis KN-104, the three kinds of bacillus subtilis 1#, withered obtained by commercial sources
Careless bacillus 2#, bacillus subtilis 3#.
3) each bacterial strain is cultivated into 10-12h in LB culture solutions (peptone 10g/L, dusty yeast 5g/L, sodium chloride 10g/L)
Bacteria suspension, with sterile bamboo stick streak inoculation on Columbia Blood Agar tablet (be purchased from Qingdao green grass or young crops medicine biology Co., Ltd),
37 DEG C of culture 18-24h, observe and dissolving circle (referring to Fig. 1) whether are formed around lawn.
From figure 1 it appears that bacillus subtilis 1#, 2#, 3# have apparent transparent circle to be formed, illustrate to give birth in thalline
Hemolysin is generated in growth process, is respectively provided with hemolytic, and the bacillus subtilis KN-104 that the present invention screens does not have hemolytic.
The culture of 2 bacillus subtilis KN-104 bacterial strains of embodiment
(1) actication of culture：
One ring Bacillus subtillis KN-104 bacterial strains of picking, are inoculated on slant medium, and 60h is cultivated in 30 DEG C,
Slant medium：Peptone 10g/L, dusty yeast 5g/L, sodium chloride 10g/L, agar powder 15g/L, pH value 7.0；
(2) seed culture：
The Bacillus subtillis KN-104 that step (1) activates is inoculated in seed culture medium, in 30 DEG C, the item of 150rpm
12h is cultivated under part,
Seed culture medium：Starch 15g/L, corn pulp 15g/L, yeast extract 3g/L, corn flour 8g/L, peptone 6g/
L, dipotassium hydrogen phosphate 0.5g/L, pH7.2；
(3) fermented and cultured：
Cultured seed liquor is accessed in fermentation medium, 40h is cultivated under conditions of 32 DEG C, 250rpm, obtains withered
Careless bacillus KN-104 zymotic fluids, work brood cell's number of the zymotic fluid reach 35,000,000,000 CFU/ml,
Fermentation medium：Starch 30g/L, corn pulp 30g/L, yeast extract 15g/L, corn flour 10g/L, peptone
25g/L, dregs of beans 15g/L, calcium chloride 0.5g/L, magnesium sulfate 0.8g/L, manganese chloride 0.2g/L, potassium dihydrogen phosphate 0.2g/L,
pH7.5。
3 bacillus subtilis KN-104 of embodiment does harm to specific test to disease-resistant crops
1) test medicine：Bacillus subtilis KN-104 fermented liquid supernatant liquid
2) for examination target：Crops Common Diseases, it is black that the present invention only lists rice blast, leaf seasonal febrile diseases, banded sclerotial blight, Chinese cabbage
Pinta, graw mold of tomato, watermelon blight, apple anthracnose, root rot, leaf muld of tomato.
3) it is measured using plating method：Take 6ml bacillus subtilises KN-104 fermented liquid supernatants liquid with it is quantitative
54ml sterilizings PDA culture medium uniformly mixes, and uniformly pours into 4 sterilising mediums respectively, and drug containing tablet is made after cooling.If without medicine
Blank control.
4) by cultured pathogen (rice blast, leaf seasonal febrile diseases, banded sclerotial blight, Cabbage Leaf Spot, graw mold of tomato, west
Cucurbit wilt, apple anthracnose, root rot, leaf muld of tomato), aseptically with sterilization punchers from colony edge
Bacteria cake (a diameter of 6mm) is cut, is trained pure culture biscuits involvng inoculation in the incubator of preference temperature in drug containing tablet center with inoculator
It supports.
5) pathogen mycelial growth situation is investigated according to the growing state of bacterium in blank control culture dish.With calliper bacterium
Fall diameter, each bacterium colony respectively once, takes its average value with crossing method vertical measurement diameter.According to investigation result, calculate each
Concentration is to the mycelial growth inhibition rate for examination target bacterium.
1 bacillus subtilis KN-104 of table does harm to specific test to disease-resistant crops
As can be seen from Table 1, bacillus subtilis KN-104 reaches the inhibiting rate of rice blast, Rice Leaf seasonal febrile diseases
100%, more than 50% is reached to the inhibiting rate of rice sheath blight disease and Cabbage Leaf Spot, show above four kinds of diseases are respectively provided with compared with
Strong rejection ability, and to the inhibition of graw mold of tomato, leaf muld of tomato, watermelon blight, apple anthracnose, root rot
Rate is very low, almost without preventive effect.
Claims (3)
1. a kind of bacillus subtilis (Bacillus subtilis) KN-104 bacterial strains, it is characterised in that be deposited in Chinese Typical Representative
Culture collection, deposit number are CCTCC NO:M2017697.
2. bacillus subtilis KN-104 bacterial strains as described in claim 1, it is characterised in that the bacterial strain is to rice blast, water
Rice leaf seasonal febrile diseases, rice sheath blight disease, Cabbage Leaf Spot have stronger rejection ability, and very low to the rejection ability of other diseases,
The fertility of the bacterial strain is strong and without hemolytic simultaneously.
3. the cultural method of bacillus subtilis KN-104 bacterial strains described in a kind of claim 1, it is characterised in that including following step
Suddenly：
(1) actication of culture：
One ring Bacillus subtillis KN-104 bacterial strains of picking, are inoculated on slant medium, and 60h is cultivated in 30 DEG C,
Slant medium：Peptone 10g/L, dusty yeast 5g/L, sodium chloride 10g/L, agar powder 15g/L, pH value 7.0；
(2) seed culture：
The Bacillus subtillis KN-104 that step (1) activates is inoculated in seed culture medium, under conditions of 30 DEG C, 150rpm
Cultivate 12h,
Seed culture medium：Starch 15g/L, corn pulp 15g/L, yeast extract 3g/L, corn flour 8g/L, peptone 6g/L, phosphorus
Sour hydrogen dipotassium 0.5g/L, pH7.2；
(3) fermented and cultured：
Cultured seed liquor is accessed in fermentation medium, 40h is cultivated under conditions of 32 DEG C, 250rpm, obtains withered grass bud
Spore bacillus KN-104 zymotic fluids, work brood cell's number of the zymotic fluid reach 35,000,000,000 CFU/ml,
Fermentation medium：Starch 30g/L, corn pulp 30g/L, yeast extract 15g/L, corn flour 10g/L, peptone 25g/L,
Dregs of beans 15g/L, calcium chloride 0.5g/L, magnesium sulfate 0.8g/L, manganese chloride 0.2g/L, potassium dihydrogen phosphate 0.2g/L, pH7.5.
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Cited By (4)
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| CN108795827A (en) * | 2018-07-06 | 2018-11-13 | 四川农业大学 | A kind of bacillus megaterium BM22 and its water dispersible granules and application |
| CN109796394A (en) * | 2018-12-27 | 2019-05-24 | 武汉科诺生物科技股份有限公司 | A method of extracting auxin from Paenibacillus polymyxa fermentation liquid |
| CN115011505A (en) * | 2022-04-24 | 2022-09-06 | 山东省农业科学院 | Bacillus subtilis and application thereof |
| CN115369062A (en) * | 2022-09-02 | 2022-11-22 | 上海市农业科学院 | Tomato bacterial wilt antagonistic bacterium WJB0802 and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795827A (en) * | 2018-07-06 | 2018-11-13 | 四川农业大学 | A kind of bacillus megaterium BM22 and its water dispersible granules and application |
| CN109796394A (en) * | 2018-12-27 | 2019-05-24 | 武汉科诺生物科技股份有限公司 | A method of extracting auxin from Paenibacillus polymyxa fermentation liquid |
| CN109796394B (en) * | 2018-12-27 | 2021-05-18 | 武汉科诺生物科技股份有限公司 | Method for extracting plant growth hormone from paenibacillus polymyxa fermentation liquor |
| CN115011505A (en) * | 2022-04-24 | 2022-09-06 | 山东省农业科学院 | Bacillus subtilis and application thereof |
| CN115011505B (en) * | 2022-04-24 | 2023-04-18 | 山东省农业科学院 | Bacillus subtilis and application thereof |
| CN115369062A (en) * | 2022-09-02 | 2022-11-22 | 上海市农业科学院 | Tomato bacterial wilt antagonistic bacterium WJB0802 and application thereof |
| CN115369062B (en) * | 2022-09-02 | 2023-10-20 | 上海市农业科学院 | Tomato bacterial wilt antagonistic bacterium WJB0802 and application thereof |
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