CN107760641A - A kind of bacillus subtilis microbial agent and its application - Google Patents
A kind of bacillus subtilis microbial agent and its application Download PDFInfo
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- CN107760641A CN107760641A CN201711303271.4A CN201711303271A CN107760641A CN 107760641 A CN107760641 A CN 107760641A CN 201711303271 A CN201711303271 A CN 201711303271A CN 107760641 A CN107760641 A CN 107760641A
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Abstract
The invention discloses a kind of microbial inoculum containing bacillus subtilis and its application.It is characterized in that it be byBacillus subtilisEBC7 fermentations are made；Fermentation medium：Soybean cake powder 15g, wheat bran 20g, K2HPO4 1.0g、NaCl 3.0g、Mg2SO4 0.5g、KNO30.06g, moisturizing to 1000mL, pH7.0 7.2.Microbial inoculum disclosed by the invention containing bacillus subtilis has efficient preventive and therapeutic effect to soil-borne disease of cucumber-cucumber root rot and damping-off.Prevention effect wherein to cucumber root rot is 82.35%；Prevention effect to damping-off is 86.77%.
Description
Technical field
The present invention relates to a kind of microbial bacterial agent, more precisely a kind of bacillus subtilis (Bacillus subtilis) EBC7 microorganism formulations and said preparation be to the preventive and therapeutic effect of soil-borne disease of cucumber-root rot and damping-off.
Background technology
Cucumber is one of China staple vegetable crop, contains the macrometabolic elements such as albumen, fat, vitamin, trace element
Matter, also containing a variety of materials with medical value such as arginine, alanine and polysaccharide, it has also become indispensable on people's dining table
One of four seasons vegetables.Cultivated area in China is more than 170 ten thousand hectares, accounts for more than the 65% of world's cucumber production area.
Cucumber rhizoctonia rot and root rot are 2 kinds of main soil-borne diseases of cucumber protecting field.The aggrieved seedling stem of cucumber rhizoctonia rot
Base portion produces ellipse or irregular shape crineous scab, and with wheel line, later stage scab expands, can be one week around stem, and sick portion hangs contracting,
Blade is wilted, the withered death of seedling.Cucumber root rot is mainly caused harm root and rhizome portion.Just it is in water soaking mode, advanced decay, rotten place
Vascular bundle browning, pink mustiness thing is produced when serious, plant is withered.
With the increase of the protecting field continuous cropping time limit, pathogen accumulates year by year, endangers getting worse.Chemical prevention is both
The main means of prevention of disease, but because pollution of the chemical pesticide to environment and agricultural product and germ easily produce to chemical agent
The resistance to the action of a drug, therefore people are sought for the method for biological control to control the occurrence and development of both diseases.
Bacillus attention always in vegetable crop disease control.Zhu Hui etc. be separated to several plants to capsicum epidemic disease and
Cucumber fusarium axysporum has the bacillus of antagonism；The bacillus subtilis microbial agent of the developments such as Zhao Changlong is to cucumber at seedling stage root
Disease-controlling effect reaches 64.12%.Because bacillus is one of constituent of soil and plant microecosystem, and
With it is safe and pollution-free the advantages of, therefore, vegetable crop protection on will play an increasingly important role.
The content of the invention
To achieve the above object, the present invention provides following technical scheme：
A kind of bacillus subtilis microbial agent, it is characterised in that it be byBacillus subtilis Made in EBC7 inoculation mediums
Into；Culture medium therein includes：NB culture mediums are：Peptone 1g, beef extract 3g, NaCl 5g, moisturizing to 1000mL, pH7.0；
Fermentation medium：The g of soybean cake powder 15, wheat bran 20 g, K2HPO4 1.0g、NaCl 3.0g、Mg2SO4 0.5g、KNO3 0.06g,
Moisturizing is to 1000mL, pH7.0-7.2.
The preparation method of bacillus subtilis microbial agent of the present invention, it is characterised in that：1 collarium has been activatedBacillus subtilis EBC7 is inoculated in the 250ml triangular flasks equipped with 100ml NB culture mediums, 37 DEG C, 200 rpm/
Min shaken cultivations 24h, 4% inoculum concentration are transferred in fermentation medium, 37 DEG C, 200 rpm/min, culture 30h, obtain produce spore
Amount reaches 4.1~6.5 × 1010Zymotic fluid；
NB culture mediums therein：Peptone 1g, beef extract 3g, NaCl 5g, moisturizing to 1000mL, pH7.0；Fermentation medium：It is yellow
The g of beancake powder 15, wheat bran 20 g, K2HPO4 1.0g、NaCl 3.0g、Mg2SO4 0.5g、KNO3 0.06g, moisturizing to 1000mL,
pH7.0-7.2。
The present invention further discloses bacillus subtilis microbial agent to the application in terms of soil-borne disease of cucumber preventing and treating.It is special
It is not to the application in terms of cucumber rhizoctonia rot and root rot preventing and treating.Experimental result is shown：The bacillus subtilis that the present invention develops
Microbial inoculum is 82.35% to the prevention effect of cucumber root rot；Prevention effect to damping-off is 86.77%.
The key point that the present invention studies is to have developed that a kind of cost is low, fermentation time is short, fermentation raw material is easy to get, fermentation is imitated
The good bacillus subtilis microbial agent of fruit, the microbial inoculum has mainly been investigated to soil-borne disease of cucumber --- the preventing and treating of root rot and damping-off
Effect.
Possessed good effect is a kind of bacillus subtilis microbial agent disclosed by the invention compared with prior art：
（1）The carbon and nitrogen sources of the bacteria fermentation are market at prices soya bean muffin and wheat bran low in cost and easily available, remaining chemical reagent
It is commercially available, and it is cheap, ensure the low cost of microbial inoculum；In addition, the bacteria fermentation time is short and easy to operate, it is economical real
With.
（2）The microbial inoculum is to being difficult to the soil-borne disease of cucumber prevented and treated --- and cucumber rhizoctonia rot and droop have good preventing and treating effect
Fruit, it is expected to solve to perplex for a long time the soil-borne disease preventing and treating problem of people.
Embodiment
Illustrate the present invention with reference to embodiment, the scheme of embodiment described here does not limit the present invention, this area it is special
Industry personnel can be made improvements and change according to the spirit of the present invention, and described such modifications and variations are regarded as at this
In the range of invention, the scope of the present invention and essence are defined by the claims.This hair is furtherd elucidate below by example
The preparation method of bright microbial bacterial agent.Reagent used in the present invention is commercially available unless otherwise indicated.Wherein bacillus subtilis
Bacterium（Bacillus subtilis）, it is in the preserving number of China Committee for Culture Collection of Microorganisms's common micro-organisms center：
CGMCC No.5586（Referring to patent ZL 201210003920.X）.
Embodiment 1
Bacillus subtilisEBC7 separation（Referring to patent ZL 201210003920.X）：
（1）From Tianjin Xiqing District greenhouse collection health, " Jinyou No.10（It is commercially available）" cucumber root；After flowing water rinses 1-2 h,
After 1 min is soaked in 70% ethanol, 6-8 min are sterilized in 2.5% NaClO, aseptic water washing is totally standby；
（2）Step sterilizing tissue obtained above is transferred in sterile mortar and adds PBS grinding, takes grinding juice system
After row dilution, respectively take 100 μ l to be coated on NA flat boards, dark culturing under the conditions of being placed in 28 DEG C, often handle 3 repetitions；
（3）Single bacterium colony on picking NA flat boards, line purifying culture, obtains Bacillus subtillis bacterial strain；Then it is stored in 15%
In glycerine -80 DEG C it is standby；NA culture mediums therein：Peptone 10g, NaCl 5g, beef extract 3g, agar powder 12g, moisturizing is extremely
1000ml.pH7.0。
Embodiment 2
A kind of bacillus subtilis microbial agent, it is characterised in that it be byBacillus subtilis Made in EBC7 inoculation mediums
Into；Culture medium therein includes：NB culture mediums：Peptone 1g, beef extract 3g, NaCl 5g, moisturizing to 1000mL, pH7.0；Hair
Ferment culture medium：The g of soybean cake powder 15, wheat bran 20 g, K2HPO4 1.0g、NaCl 3.0g、Mg2SO4 0.5g、KNO3 0.06g, mend
Water is to 1000mL, pH7.0-7.2.
The preparation method of bacillus subtilis microbial agent：1 collarium has been activatedBacillus subtilis EBC7 is inoculated with
In the 250ml triangular flasks equipped with 100ml NB culture mediums, 37 DEG C, 200 rpm/min shaken cultivations 24h, 4% inoculum concentration turn
Be connected in fermentation medium, 37 DEG C, 200 rpm/min, culture 30h, obtain sporulation quantity and reach 4.1~6.5 × 1010Fermentation
Liquid；NB culture mediums therein：Peptone 1g, beef extract 3g, NaCl 5g, moisturizing to 1000 mL, pH7.0；Fermentation medium：It is yellow
The g of beancake powder 15, wheat bran 20 g, K2HPO4 1.0g、NaCl 3.0g、Mg2SO4 0.5g、KNO3 0.06g, moisturizing to 1000mL,
pH7.0-7.2。
Test examples 1.
Inhibitory action of the microbial bacterial agent to pathogen
By the Fusarium solani (Agricultural University Of Hebei's offer) of activation and Rhizoctonia solani Kuhn（Agricultural University Of Hebei provides）It is inoculated into
On PDA plate, 25 DEG C of culture 7d, pure culture biscuits involvng inoculation is taken in fresh PDA plate side in colony edge with Φ=3.5mm card punch,
Φ=3.5mm aseptic filter paper piece is placed on its opposite, 10 μ l are inoculated with thereonBacillus subtilisEBC7 zymotic fluids,
10 μ l sterilized waters replace zymocyte liquid as control.Colony radius are measured after 7 days, and calculate bacteriostasis rate.
Inhibitory action of the microbial bacterial agent of table 1. to cucumber pathogen
Test examples 2
Preventive and therapeutic effect of the microbial bacterial agent to cucumber root rot
The Fusarium solani of activation is inoculated on fresh PDA flat board, 25 DEG C of culture 7d, is added into corn sand culture medium,
25 DEG C of 10 d of culture.Above-mentioned culture is smashed, with sterilization soil according to 1:50 (W/W) are mixed, and dress alms bowl (20cm × 20cm) is standby.
No. 4 cucumber seeds are ground with 500 × dilution of microbial bacterial agent according to 20 in Tianjin:1 (W/W) dresses seed, and is sowed in alms bowl, per 10 kinds of basin
Son.4 alms bowls are often handled, experiment is repeated 3 times.Clear water seed dressing is control, and sowing investigates emergence rate after 7 days, and diseased plant rate is investigated at 15 days
Incidence, and calculate disease index and relative prevention effect.Sick level is as follows：0 grade is not fallen ill；1 grade of root or basal part of stem scab
Area accounts for the 1%-10% of the gross area；2 grades of roots or basal part of stem lesion area account for 11%-30%；3 grades of roots or basal part of stem lesion area
Account for 31%-60%；4 grades of roots or basal part of stem lesion area account for more than 61%.Using SPSS statistical softwares, using single factor test variance ratio compared with
（LSD）Significance analysis is carried out to result of the test.
Prevention effect of the probiotics of table 2. to cucumber root rot
Note：Same column data acceptance of the bid has significant difference (P between different alphabetical expression processing<0.01)
Table 2 shows that after microbial bacterial agent processing, cucumber emergence rate significantly improves, and reaches 91.67%；Under diseased plant rate is notable
Drop, only 6.36%；Disease index significantly reduces, and is 6.82；82.38% is reached to the relative prevention effect of cucumber root rot.
Test examples 3
Preventive and therapeutic effect of the microbial bacterial agent to cucumber rhizoctonia rot
Rhizoctonia solani Kuhn is gone into PDA culture medium x to be activated, 25 DEG C of culture 7d, is transferred in wheat sand culture medium, 25
DEG C culture 20 days.Culture is smashed, with 3:100 (W/W) ratios mix with sterile soil, and dress alms bowl (20cm × 20cm) is standby.Tianjin
No. 4 cucumber seeds are ground with 500 × dilution of microbial bacterial agent according to 20:1 (W/W) dresses seed, and is sowed in alms bowl, per 10 seeds of basin.
4 alms bowls are often handled, experiment is repeated 3 times.Clear water seed dressing is control, and sowing investigates emergence rate after 7 days, diseased plant rate investigation morbidity at 15 days
Situation, and calculate disease index and relative prevention effect.Sick level is as follows：0 grade is not fallen ill；1 grade of root or basal part of stem lesion area
Account for the 1%-25% of the gross area；2 grades of roots or basal part of stem lesion area account for 26%-50%；3 grades of roots or basal part of stem lesion area account for
51%-75%；4 grades of roots or basal part of stem lesion area account for more than 75%.Using SPSS statistical softwares, using single factor test variance ratio compared with
（LSD）Significance analysis is carried out to result of the test.
Prevention effect of the probiotics of table 3. to cucumber rhizoctonia rot
Note：Same column data acceptance of the bid has significant difference (P between different alphabetical expression processing<0.01)
Table 3 shows that after probiotics processing, cucumber emergence rate significantly improves, and reaches 93.33%；Diseased plant rate and the state of an illness refer to
Number significantly reduces than control, and respectively 5.36% and 4.33；Reach 86.77% with respect to prevention effect.
Claims (4)
- A kind of 1. bacillus subtilis microbial agent, it is characterised in that it be byBacillus subtilis EBC7 inoculation mediums are sent out Ferment is made；Fermentation medium：The g of soybean cake powder 15, wheat bran 20 g, K2HPO4 1.0g、NaCl 3.0g、Mg2SO4 0.5g、KNO3 0.06g, moisturizing to 1000mL, pH7.0-7.2.
- 2. the preparation method of the bacillus subtilis microbial agent described in claim 1, it is characterised in that：1 collarium has been activatedBacillus subtilis EBC7 is inoculated in the 250ml triangular flasks equipped with 100ml NB culture mediums, 37 DEG C, 200 rpm/ Min shaken cultivations 24h, 4% inoculum concentration are transferred in fermentation medium, 37 DEG C, 200 rpm/min, culture 30h, obtain produce spore Amount reaches 4.1~6.5 × 1010Zymotic fluid；Fermentation medium therein is：The g of soybean cake powder 15, wheat bran 20 g, K2HPO4 1.0g、NaCl 3.0g、Mg2SO4 0.5g、KNO3 0.06g, moisturizing to 1000 mL, pH7.0-7.2.
- 3. bacillus subtilis microbial agent described in claim 1 is to the application in terms of cucumber root rot preventing and treating.
- 4. microbial bacterial agent described in claim 1 is to the application in terms of cucumber rhizoctonia rot preventing and treating.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113303340A (en) * | 2021-05-21 | 2021-08-27 | 宁夏大学 | Composition of biocontrol bacteria and fish protein and application of composition in cucumber cultivation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1237346A (en) * | 1999-06-15 | 1999-12-08 | 南京农业大学 | Bacillus subtilis preparation, production method and its application |
| CN102533579A (en) * | 2010-12-30 | 2012-07-04 | 中国科学院沈阳应用生态研究所 | Culture medium for producing spores, and optimizing process and application thereof |
| CN102586136A (en) * | 2012-01-09 | 2012-07-18 | 天津市农业生物技术研究中心 | Microbial inoculant for improving disease resistance and promoting growth of plants |
-
2017
- 2017-12-11 CN CN201711303271.4A patent/CN107760641A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1237346A (en) * | 1999-06-15 | 1999-12-08 | 南京农业大学 | Bacillus subtilis preparation, production method and its application |
| CN102533579A (en) * | 2010-12-30 | 2012-07-04 | 中国科学院沈阳应用生态研究所 | Culture medium for producing spores, and optimizing process and application thereof |
| CN102586136A (en) * | 2012-01-09 | 2012-07-18 | 天津市农业生物技术研究中心 | Microbial inoculant for improving disease resistance and promoting growth of plants |
Non-Patent Citations (1)
| Title |
|---|
| 李达 等: "饲用枯草芽孢杆菌高密度发酵培养基的优化", 《东北农业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113303340A (en) * | 2021-05-21 | 2021-08-27 | 宁夏大学 | Composition of biocontrol bacteria and fish protein and application of composition in cucumber cultivation |
| CN113303340B (en) * | 2021-05-21 | 2022-05-31 | 宁夏大学 | Composition of biocontrol bacteria and fish protein and application of composition in cucumber cultivation |
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Application publication date: 20180306 |