CN109468242A - The Bacillus strain and microbial inoculum and bacterial preparation process of a kind of two kinds of diseases of prevention and treatment and application - Google Patents
The Bacillus strain and microbial inoculum and bacterial preparation process of a kind of two kinds of diseases of prevention and treatment and application Download PDFInfo
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Abstract
The invention discloses a kind of Bacillus strain of two kinds of diseases of prevention and treatment and microbial inoculum and bacterial preparation process and applications, the Bacillus strain of two kinds of diseases of prevention and treatment is bacillus amyloliquefaciens CJ20, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation day: on October 11st, 2012, deposit number: CGMCC No.6666.The microbial inoculum of the bacillus amyloliquefaciens CJ20 is the microbial inoculum being prepared with the bacillus amyloliquefaciens CJ20 through inclined-plane culture, seed culture and fermentation.Preparation method includes inclined-plane culture, seed culture, fermentation and microbial inoculum preparation step；The application in prevention and treatment tobacco root-knot and tobacco black shank drug is used to prepare using the microbial inoculum for the bacillus amyloliquefaciens CJ20.The bacillus amyloliquefaciens CJ20 is that the preparation of active constituent all has significant preventive and therapeutic effect to tobacco black shank and root-knot nematode.
Description
Technical field
The invention belongs to microorganisms to be separately cultured technical field, and in particular to a kind of bacillus bacterium for preventing and treating two kinds of diseases
Strain and microbial inoculum and bacterial preparation process and application.
Background technique
Two kinds of main soil-borne diseases on tobacco black shank and root-knot nematode medical history tobacco.With the development of tobacco leaf production,
The increase of continuous cropping area, harm are on the rise, and nematodiasis is one of the key constraints of many countries tobacco productions, at me
The major cigarette district of state causes serious harm, and diseased plant causes tobacco leaf underproduction 30%-50%.Tobacco black shank is Breda de in 1896
A kind of silborne fungal diseases that Haan has found on Indonesia Java tobacco, mostly occur in Adult plant, also there is a small number of seedbed phases.
The disease is the destructive disease of tobacco, each Chan Yan state of throughout world.In China, in addition to colder Heilongjiang Province, other are each
Producing cigarette province has different degrees of generation, and harm is more universal and serious, and disease incidence is generally in 10%-15%, average annual economy damage
It loses up to 100,000,000 yuans or more.
In recent years since continuous cropping tobacco field area expands and the growth of the continuous cropping time limit, many place tobacco black shanks and root knot
Nematodiasis has the tendency that aggravating popular.Due to lacking ideal disease-resistant tobacco cultivars in production, currently, tobacco black shank
Prevention and treatment with root knot nematode disease still relies on chemical agent, but effect is not satisfactory, and there are many drawbacks.As used at present
Chemical nematicides be mostly severe toxicity or high poison, persistent pesticide, edaphon, plant, water source and atmosphere are brought sternly
Heavily contaminated or destruction, while resistance problems also become increasingly conspicuous, and drug is remained in plant and can be caused indirectly to the mankind
Harm.Therefore it is badly in need of developing environmentally friendly, nontoxic to people and animals microorganism formulation.
What the effective way and recent years that plant pest is prevented and treated using the antagonistic bacterium grown nonparasitically upon another plant on plant were risen
New research hotspot, and screening efficiently is then the prerequisite item for guaranteeing biological control and succeeding with activity stabilized antagonistic strain
Part.At present the biocontrol bacteria mainly studied have plant growth-promoting rhizobacteria (PGPR), puncture pasteurella (Pasteuria penetrans), Pseudomonas alba (Pseudomonas spp.), bacillus thuringiensis (Bacillus thuringiensis) etc..Endophytic bacterium is largely present in the plant tissue of health and illness, these plant endogenesis are thin
Bacterium can colonize between plant tissue, and living environment is stablized, and is not easily susceptible to the influence of external environment, and to people and animals' nonhazardous, right
It is environmental-friendly, it is important plant disease biocontrol bacteria resource, is had become closely as new bio-control factors controlling plant diseases
The big hot spot of the one of microbiological research in 10 years shows endogenetic bacteria and is used as to the non-harmful biological pesticide development and application of environment
Good prospect.
Summary of the invention
The first object of the present invention is to provide a kind of Bacillus strain-solution starch gemma bar for preventing and treating two kinds of diseases
Bacterium (Bacillus amyloliquefaciens) CJ20；Second is designed to provide the bacillus amyloliquefaciens
(Bacillus amyloliquefaciens) CJ20 microbial inoculum；Third is designed to provide the bacillus amyloliquefaciens
(Bacillus amyloliquefaciens) CJ20 microbial inoculum preparation method；4th is designed to provide the solution starch
Bacillus (Bacillus amyloliquefaciens) CJ20 microbial inoculum application.
Outer unless otherwise indicated, percentage employed in the present invention is percentage by volume.
The first object of the present invention is achieved in that the Bacillus strain of two kinds of diseases of prevention and treatment for solution starch
Bacillus (Bacillus amyloliquefaciens) CJ20, depositary institution: Chinese microorganism strain preservation conservator
Meeting common micro-organisms center (CGMCC), preservation day: on October 11st, 2012, deposit number: CGMCC No.6666, the Xie Dian
Afnyloliquefaciens belong to eubacteria circle, rear wall bacterium door, bacillus guiding principle, bacillus head, Bacillaceae, bacillus.
--- bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 acquisition and identification
(1) bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 acquisition and identification
Bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) CJ20, it is from tobacco stalk
It is isolated.Tobacco sample picks up from the Yunnan Province town Yuxi Yan He, acquires the disease-free cigarette strain prosperous growth phase stalk that grows fine, after cleaning
1min is impregnated in oscillation in 70% alcohol, then with after liquor natrii hypochloritis's oscillation immersion 1min of available chlorine content 1%, uses sterile water
It rinses 4 times.Stem tissue and sterilizing steel ball after disinfection are put into 2ml sterile centrifugation tube together, and 600ul sterile water is added, uses
QIAGEN high-flux tissue dismembyator grinds 3min, frequency 30r/s.Mother liquor after grinding is applied after being diluted using gradient dilution method
Cloth plate, the dark culturing 48h at 28 DEG C.Picking single colonie, again in flat lining out purification storage, wherein growing way is vigorous
Person names as CJ20 bacterial strain.
Used medium formula: Yeast diffusion juice 3.0g, beef extract 1.0g, soy peptone 5.0g, NaCl 5.0g,
Agar 17.0g, distilled water 1000mL, pH:7.0.
To above-mentioned isolated strains B. amyloliquefaciens (Bacillus amyloliquefaciens) CJ20, pass through life
Object characteristic and morphologic observation carry out Identification of Species, the method is as follows:
Form, the size, camber, edge of bacterium colony, Gram's staining are observed, physiological and biochemical property tests specific method reference
" common bacteria system identification handbook ".The bacterium 16S rDNA sequence amplification primer uses F27/R1492, and amplified production entrusts Shanghai
Ying Jun Bioisystech Co., Ltd carries out sequencing.
Thallus shape be it is rod-shaped, thallus mean size be 0.5 ~ 1.5um × 1.6 ~ 2.5um, Gram's staining be the positive.It is interior
It sprouts spore, atrichia.Bacterium colony is white on NA culture medium, fold protrusion, and edge is irregular.Aerobic growth, glucose fermentation
The positive, Starch Hydrolysis test is positive, and gelatin liquefaction test is positive, and V.P test is positive, catalase reacting positive, clark and Lubsreaction
The positive, nitrate reduction test is positive, and citrate utilizes the positive.At 8 ~ 45 DEG C, optimum is grown the bacterium growth temperature range
Temperature is 29 ~ 33 DEG C, and for growth pH value 6.5 ~ 8, optimum pH value is 7.
The physiological and biochemical property of 1 bacterial strain CJ20 of table
Note: "+" indicates positive reaction, and "-" indicates negative reaction.
By the morphological feature of bacterial strain, cultural characteristic, physio-biochemical characteristics meet bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain characteristic.
The analysis of 16S rDNA sequence: 16S rDNA sequencing is carried out to the bacterial strain, obtains the nucleic acid sequence of 1514bp length
Column, GenBank number of registration: JQ936678.1.CJ20 bacterial strain 16S rDNA sequence and the bacterial strain similitude of bacillus reach
99%.By sequence alignment and physio-biochemical characteristics by CJ20 bacterial strain be accredited as bacillus amyloliquefaciens (Bacillus amyloliquefaciens).Its 16S rDNA sequence of CJ20 bacterial strain is shown in GenBank:JQ936678.1.The solution starch gemma bar
Pseudomonas is in eubacteria circle, rear wall bacterium door, bacillus guiding principle, bacillus head, Bacillaceae, bacillus.
(2) bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 preservation
By above-mentioned qualification result, confirmation bacterial strain CJ20 be bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
A bacterial strain, be named as CJ20, it is common to be preserved in China Committee for Culture Collection of Microorganisms on October 11st, 2012
Microorganism center (abbreviation CGMCC, address are as follows: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode
100080), deposit number is CGMCC No.6666.
The second object of the present invention be achieved in that with the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 is prepared through inclined-plane culture, seed culture, fermentation and microbial inoculum.
The third object of the present invention is achieved in that prepare including inclined-plane culture, seed culture, fermentation and microbial inoculum and walk
Suddenly, it specifically includes:
A, inclined-plane culture: by bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 is inoculated into inclined-plane culture
On base, 48h is cultivated at being 28 ~ 30 DEG C in temperature, obtains slant strains；
B, seed culture: slant strains are inoculated into fluid nutrient medium, temperature be 29 ~ 33 DEG C at culture 18 ~ for 24 hours, obtain liquid
Body fermentation seed；
C, it ferments: liquid fermentation seed being inoculated with by the amount of fermentation medium volume 3 ~ 5% into fermentor, is cultivated at 29 ~ 33 DEG C
36 ~ 48h, mixing speed be 120 ~ 150rpm, ventilatory capacity 5 ~ 10%, obtain bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 fermentation liquid；
D, prepared by microbial inoculum: the activating agent of addition 0.16 ~ 0.6% mixes well in fermentation liquid, obtains object microbial inoculum.
The fourth object of the present invention be achieved in that the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 microbial inoculum be used to prepare prevention and treatment tobacco root-knot and tobacco black shank drug in answering
With.
It bacillus amyloliquefaciens of the present invention and can be applied by the preparation of active constituent of the bacillus amyloliquefaciens
In the prevention and treatment of tobacco black shank and root-knot nematode, have the advantage that
1, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 is a kind of efficient biocontrol bacteria, it is with it
The preparation of active constituent all has significant preventive and therapeutic effect to tobacco black shank and root-knot nematode.
2, preparation method is simple for preparation, rationally effectively, it is low in cost, be conducive to said preparation industrialized production and
Application popularization.
3, the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 and with its for activity at
The preparation divided is nontoxic to people and animals, and has very strong environmental suitability and environment friendly.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated, but the present invention is limited in any way,
Based on present invention teach that it is made it is any transform or replace, all belong to the scope of protection of the present invention.
It is of the present invention prevention and treatment two kinds of diseases Bacillus strain be bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC), preservation day: on October 11st, 2012, deposit number: CGMCC No.6666, the bacillus amyloliquefaciens belong to very
Bacterium circle, rear wall bacterium door, bacillus guiding principle, bacillus head, Bacillaceae, bacillus.
Classification naming: bacillus amyloliquefaciens CJ20
Latin literary fame:Bacillus amyloliquefaciens
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Address: Institute of Microorganism, Academia Sinica, BeiJing, China
Preservation date: on October 11st, 2012
Deposit number: CGMCC NO:6666.
Bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) CJ20 microbial inoculum, be with
The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 is through inclined-plane culture, seed culture, fermentation
It is prepared with microbial inoculum.
Bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) CJ20 microbial inoculum system
Preparation Method, including inclined-plane culture, seed culture, fermentation and microbial inoculum preparation step, specifically include:
A, inclined-plane culture: by bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 is inoculated into inclined-plane culture
On base, 48h is cultivated at being 28 ~ 30 DEG C in temperature, obtains slant strains；
B, seed culture: slant strains are inoculated into fluid nutrient medium, temperature be 29 ~ 33 DEG C at culture 18 ~ for 24 hours, obtain liquid
Body fermentation seed；
C, it ferments: liquid fermentation seed being inoculated with by the amount of fermentation medium volume 3 ~ 5% into fermentor, is cultivated at 29 ~ 33 DEG C
36 ~ 48h, mixing speed be 120 ~ 150rpm, ventilatory capacity 5 ~ 10%, obtain bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 fermentation liquid；
D, prepared by microbial inoculum: the activating agent of addition 0.16 ~ 0.6% mixes well in fermentation liquid, obtains object microbial inoculum.
Slant medium described in step A is nutrient agar.
Fluid nutrient medium described in step B is beef extract 2 ~ 4%, the peptone 0.4 ~ 0.6%, chlorination of mass percent
Sodium 0.4 ~ 0.6%, remaining is water, and pH value is 6.5 ~ 8.0.
Fermentation medium described in step C is beancake powder 1 ~ 2%, soluble starch 1 ~ 2%, the beef leaching of mass percent
Cream 0.5 ~ 0.8%, NaCNO30.02~0.04%、CaCO30.1 ~ 0.3%, remaining is water.
Activating agent described in D step be the sodium humate of percent by volume 0.05 ~ 0.2%, percent by volume 0.01 ~
One or more of 0.1% chitosan and the chitin of percent by volume 0.1 ~ 0.3%.
Bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) microbial inoculum of CJ20 answers
With for the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) microbial inoculum of CJ20 is used to prepare prevention and treatment cigarette
Application in grass roots Root-knot and tobacco black shank drug.
Case is embodied, the present invention will be further described below:
Embodiment 1
--- the prevention and treatment of root knot nematode in tobacco is tested with bacillus amyloliquefaciens CJ20 body preparation
The separation of root-knot nematode is carried out using the graceful funnel method of shellfish, obtains the second instar larvae of root-knot nematode.By isolated root-knot nematode
It is added in 100 times of dilutions of bacillus amyloliquefaciens liquid preparation, is control with the water of equivalent, is placed at room temperature for the 1h line of observation
Worm survival and The dead quantity, the results are shown in Table 2.The result shows that the death rate of root-knot nematode second instar larvae is 8.5% in control,
And the death rate of the processing root-knot nematode second instar larvae of 100 times of dilutions of bacillus amyloliquefaciens liquid preparation reaches
96.6%, it can be seen that, have using bacillus amyloliquefaciens CJ20 as the liquid preparation of active constituent to root-knot nematode second instar larvae
There is strong toxic effect.
Toxic effect of the 2 bacillus amyloliquefaciens liquid preparation of table to root-knot nematode
| Treatment fluid | Borer population (item) living | Dead borer population (item) | The death rate (%) |
| Water | 65 | 6 | 8.5 |
| CJ20 liquid preparation | 3 | 85 | 96.6 |
Flue-cured tobacco cultivars are Hongda tobacco, and root-knot nematode vaccination ways are sick soil inoculation, and every plant of cigarette applies Xie Dian when flue-cured tobacco transplanting
Afnyloliquefaciens preparation 20ml, not apply preparation as control.It uproots within 60 days after transplanting and investigates the state of an illness of tobacco root-knot
Index the results are shown in Table 3.It is 21.3 by the tobacco root-knot feelings index that bacillus amyloliquefaciens preparation is handled, without
Application said preparation blank control disease index reached 70.2, it can be seen that, with bacillus amyloliquefaciens CJ20 be activity at
The preparation divided is 69.7% to the control efficiency of tobacco root-knot.
Control efficiency of the 3 bacillus amyloliquefaciens preparation of table to root knot nematode disease
| Processing | Disease index | Control efficiency (%) |
| CK | 70.2 | ? |
| CJ20 preparation | 21.3 | 69.7 |
Embodiment 2
--- bacillus amyloliquefaciens CJ20 preparation tests the prevention and treatment of tobacco black shank
The preparation of cause of disease inoculum: by Phytophthora nicotianae transplanting in CA plate, 7d is cultivated in 28 DEG C of inversions.Ferfas silk block is taken to be inoculated into
In oat culture solution, 28 DEG C, 180r/min culture 7d after be transferred on bacterium paddy culture medium cultivate 21d it is spare.
Bacillus amyloliquefaciens CJ20 is prepared by the liquid preparation of active constituent: with A, B, C and D in preparation.
Prevention and treatment experiment: after tobacco seedlings transplanting goes back seedling 3 days, endogenetic bacteria is inoculated with using root-pouring method.It is wet with a small amount of clear water before pouring root
Moisten soil, antagonistic strain suspension is diluted 5 times, every plant of pouring root 10ml, control is handled with NB culture solution.Pouring root 1 time every three days,
Pouring root 5 times altogether.It is inoculated with black shank bacterium after inoculation endogenetic bacteria 7 days, first scratches cigarette strain basal part of stem with scalpel, then inoculation training
Phytophthora nicotianae Breda paddy is supported, inoculum concentration is 2g/ plants, and a water is respectively poured in earthing moisturizing immediately after inoculation sooner or later daily.Every processing 15
Strain investigates incidence after being inoculated with 14d.Tobacco black shank grade scale is referring to People's Republic of China's tobacco business standard (GB/
T 23222-2008) regulation execution.
Experimental result: Disease investigation is each to handle different journeys the 14th day after being inoculated with Phytophthora nicotianae the result shows that (table 4)
The morbidity of degree ground, endogenetic bacteria CJ20 bacterium solution pouring processing show certain protection effect, disease index 33.3, lower than control
NB cultivates liquid irrigating root, and preventive effect is respectively 46.6%, has preferable application value.
4 bacillus amyloliquefaciens preparation of table is to tobacco black shank control efficiency
Claims (8)
1. the Bacillus strain of one plant of prevention and treatment, two kinds of diseases, it is characterised in that the bacillus bacterium of described two kinds of diseases of prevention and treatment
Strain be bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20, depositary institution: Chinese microorganism strain is protected
It hides administration committee's common micro-organisms center (CGMCC), preservation day: on October 11st, 2012, deposit number: CGMCC
No.6666, the bacillus amyloliquefaciens belong to eubacteria circle, rear wall bacterium door, bacillus guiding principle, bacillus head, Bacillaceae,
Bacillus.
2. a kind of bacillus amyloliquefaciens described in claim 1 (Bacillus amyloliquefaciens) CJ20 bacterium
Agent, it is characterised in that be with the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 trains through inclined-plane
Feeding, seed culture, fermentation and microbial inoculum are prepared.
3. a kind of bacillus amyloliquefaciens as claimed in claim 2 (Bacillus amyloliquefaciens) CJ20 microbial inoculum
Preparation method, it is characterised in that including inclined-plane culture, seed culture, fermentation and microbial inoculum preparation step, specifically include:
A, inclined-plane culture: by bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 is inoculated into inclined-plane culture
On base, 48h is cultivated at being 28 ~ 30 DEG C in temperature, obtains slant strains；
B, seed culture: slant strains are inoculated into fluid nutrient medium, temperature be 29 ~ 33 DEG C at culture 18 ~ for 24 hours, obtain liquid
Body fermentation seed；
C, it ferments: liquid fermentation seed being inoculated with by the amount of fermentation medium volume 3 ~ 5% into fermentor, is cultivated at 29 ~ 33 DEG C
36 ~ 48h, mixing speed be 120 ~ 150rpm, ventilatory capacity 5 ~ 10%, obtain bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 fermentation liquid；
D, prepared by microbial inoculum: the activating agent of addition 0.16 ~ 0.6% mixes well in fermentation liquid, obtains object microbial inoculum.
4. preparation method according to claim 3, it is characterised in that slant medium described in step A is nutrient agar
Culture medium.
5. preparation method according to claim 3, it is characterised in that fluid nutrient medium described in step B is quality percentage
Beef extract 2 ~ 4%, peptone 0.4 ~ 0.6%, the sodium chloride 0.4 ~ 0.6% of ratio, remaining is water, and pH value is 6.5 ~ 8.0.
6. preparation method according to claim 3, it is characterised in that fermentation medium described in step C is quality percentage
Beancake powder 1 ~ 2%, soluble starch 1 ~ 2%, the beef extract 0.5 ~ 0.8%, NaCNO of ratio30.02~0.04%、CaCO30.1 ~ 0.3%,
Remaining is water.
7. preparation method according to claim 3, it is characterised in that activating agent described in D step is percent by volume
In the chitin of 0.05 ~ 0.2% sodium humate, the chitosan of percent by volume 0.01 ~ 0.1% and percent by volume 0.1 ~ 0.3%
One or more.
8. a kind of bacillus amyloliquefaciens as claimed in claim 2 (Bacillus amyloliquefaciens) CJ20 microbial inoculum
Application, it is characterised in that the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CJ20 microbial inoculum use
Application in preparation prevention and treatment tobacco root-knot and tobacco black shank drug.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111849839A (en) * | 2020-08-11 | 2020-10-30 | 江西顺泉生物科技有限公司 | Preparation and application of two composite bacterial liquids for preventing and treating root knot nematode disease |
| CN112341284A (en) * | 2020-11-24 | 2021-02-09 | 商洛学院 | Special microbial fertilizer for tobacco and preparation method and application thereof |
| CN112694990A (en) * | 2020-12-25 | 2021-04-23 | 中农绿康（北京）生物技术有限公司 | Bacillus amyloliquefaciens and application thereof in tomato nematode disease control |
| CN114365749A (en) * | 2022-01-27 | 2022-04-19 | 云南农业大学 | Composition for preventing and treating black rot and black shank of tobacco root and improving character and quality |
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| CN111849839A (en) * | 2020-08-11 | 2020-10-30 | 江西顺泉生物科技有限公司 | Preparation and application of two composite bacterial liquids for preventing and treating root knot nematode disease |
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| CN114365749A (en) * | 2022-01-27 | 2022-04-19 | 云南农业大学 | Composition for preventing and treating black rot and black shank of tobacco root and improving character and quality |
| CN114365749B (en) * | 2022-01-27 | 2022-12-23 | 云南农业大学 | Composition for preventing and treating black rot and black shank of tobacco root and improving character and quality |
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