CN113773979A - Preparation and application of active bacterium Bacillus sp for inhibiting tobacco mildew - Google Patents
Preparation and application of active bacterium Bacillus sp for inhibiting tobacco mildew Download PDFInfo
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- CN113773979A CN113773979A CN202110842361.0A CN202110842361A CN113773979A CN 113773979 A CN113773979 A CN 113773979A CN 202110842361 A CN202110842361 A CN 202110842361A CN 113773979 A CN113773979 A CN 113773979A
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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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
- C12N1/14—Fungi; Culture media therefor
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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
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Abstract
The invention discloses preparation and application of an active bacterium Bacillus sp for inhibiting tobacco mildew and rot, relating to the technical field of microbial pesticides, and the preparation process of the active bacterium Bacillus sp for inhibiting tobacco mildew and rot comprises the following steps: NL-6 bacteria were inoculated into tube LB liquid medium (5 ml per tube) with the following formulation: 0.5% yeast extract, 1% peptone; 1% NaCl. NL-6 cells were inoculated onto a medium and cultured by shaking at 37 ℃ for 12 hours at a rotation speed of 220rpm to obtain test tube seeds. The Bacillus sp strain has inhibitory activity on various pathogenic bacteria of the tobacco mildew rot, and is determined as Bacillus sp spore bacteria by performing morphological, physiological, biochemical and molecular identification on the strain after determining the activity of the pathogenic bacteria of the tobacco mildew rot through a plurality of batches of activity tests of the pathogenic bacteria of the tobacco mildew rot.
Description
Technical Field
The invention relates to the technical field of microbial pesticides, in particular to preparation and application of an active bacterium Bacillus sp for inhibiting tobacco mildew and rot.
Background
The tobacco leaves are the most important raw materials for producing cigarette products, and the tobacco leaves are stored for at least more than 1-2 years in the storage process from purchasing and warehousing, threshing and redrying to producing the cigarette products, so that the mildew can cause great influence on the storage of the tobacco leaves, and even the tobacco leaves partially or completely lose the use value. The longer the tobacco leaves are stored, the greater the likelihood of mildew occurring. The mildew and deterioration of the tobacco leaves are caused by the fact that the tobacco leaves are infected by the mildew, the mildew exists in the air of the nature and on the tobacco leaves and tobacco shreds, and when the environmental conditions are proper, the mildew absorbs nutrients such as sugar, moisture and protein of the tobacco leaves to grow and propagate, so that the tobacco leaves are mildewed.
The research work of China on the aspects of smoke storage and mould prevention starts late, the scientific research and technical strength are relatively weak, and necessary theoretical and technical support cannot be provided for smoke storage and mould prevention of China. At present, although the methods can be controlled through physical and chemical prevention and treatment, the methods have certain limitations and disadvantages, the operability and adaptability are not ideal, and the application of chemical drug agents is gradually limited along with the enhancement of environmental awareness of people in recent years. Therefore, the research on the biocontrol of the tobacco mildew rot is naturally an important and focused problem.
Disclosure of Invention
The invention aims to provide preparation and application of an active bacterium Bacillus sp for inhibiting tobacco mildew and rot, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
the preparation and application of an active bacterium Bacillus sp for inhibiting the tobacco mildew rot disease are as follows: NL-6 bacteria were inoculated into tube LB liquid medium (5 ml per tube) with the following formulation: 0.5% yeast extract, 1% peptone; 1% NaCl. Inoculating NL-6 thalli to a culture medium, and carrying out shake culture at 37 ℃ for 12h at the rotating speed of 220rpm to obtain test tube seeds;
the preparation process of the active bacterium Bacillus sp for inhibiting the tobacco mold rot comprises the steps of transferring test tube seeds into a fresh LB liquid culture medium test tube, and carrying out shake culture at 37 ℃ for 12 hours at the rotating speed of 220rpm to obtain a bacterium solution with strong activity;
100uL of the bacterial liquid is taken to be coated on a fresh LB solid culture medium and cultured in a constant temperature box at 37 ℃ for 12 hours to obtain the Petri dish bacteria.
As a further scheme of the invention: the preparation process of the active bacterium Bacillus sp for inhibiting the tobacco mildew rot also comprises the following steps of inoculating the test tube strain into a 250ml triangular flask liquid culture medium (100 ml per bottle), wherein the formula of the liquid culture medium is as follows: 0.5% yeast extract; 1% peptone; the remainder of 1% NaCl was water. Culturing at 37 deg.C for 24h with shaking at 220 rpm;
the fermentation culture was centrifuged at 8500rpm/min for 20 minutes, and the supernatant was collected and filtered through a 0.22um filter to obtain a sterile fermentation broth.
As a further scheme of the invention: also comprises the following preparation steps;
the method comprises the following steps: the test tube species culture is carried out, and the formula of a culture medium is as follows: 0.5% yeast extract, 1% peptone; 1% NaCl; the rest is water. Inoculating the NL-6 bacillus thalli to a culture medium, and culturing at 37 ℃ for 12h to obtain test tube seeds;
step two: liquid amplification culture, the formula of the liquid culture medium is as follows: 0.5% yeast extract; 1% peptone; 1% NaCl; the rest is water. Inoculating test tube seeds into 500ml of triangular flask liquid culture medium, wherein 200ml of each bottle is filled, and performing shake culture at 37 ℃ for 12h at the rotating speed of 220 rpm;
step three: the formula of the culture medium is as follows: 0.5% yeast extract, 1% peptone; 1% NaCl; 1.5-2% agar. Inoculating the NL-6 bacillus thallus to a culture medium, and culturing at 37 ℃ for 12h to obtain a plate strain.
As a further scheme of the invention: the method also comprises a test of inhibiting the tobacco mould rotting pathogenic bacteria by the NL-6 bacteria, and specifically comprises the following procedures;
the first process is as follows: preparing test bacteria liquid, fungus cakes and the like, inoculating and culturing the NL-6 strain according to the liquid amplification culture method and a petri dish, and preparing the bacteria liquid and the fungus cakes according to the method for inhibiting spore germination and hypha growth of the tobacco mildew and rot pathogenic bacteria.
And preparing a contrast reagent, wherein LB with corresponding amount is adopted as a blank contrast in the test for measuring the inhibition of the NL-6 bacteria on the hypha growth of the tobacco mildew-rot pathogenic bacteria and the test for the spore germination of the fermentation liquor of the NL-6 bacteria on the tobacco mildew-rot pathogenic bacteria.
And thirdly, preparing the tobacco mould-rotting pathogenic bacteria flat plate and the tobacco mould-rotting pathogenic bacteria spores for the test.
As a further scheme of the invention: the method also comprises the steps of preparing a tobacco mould and rot pathogen bacterium cake, taking down the tobacco mould and rot pathogen bacterium with good growth state from an original plate by using a puncher with the diameter of 1cm, inoculating the tobacco mould and rot pathogen bacterium cake into a new PDA solid plate in a super clean workbench, and culturing the PDA solid plate at the constant temperature of 25 ℃ until the diameter of a bacterial colony is 3cm for later use.
As a further scheme of the invention: the method further comprises the steps of preparing a spore suspension of the tobacco mildew-rot pathogenic bacteria, inoculating a spore bacterial liquid of the tobacco mildew-rot pathogenic bacteria into a Czochralski liquid culture medium in a 100ml triangular flask according to the proportion of 1:100, and culturing at the constant temperature of 25 ℃ for 180r/min until the spore concentration is 1 × 106-1 × 107cfu/ml for later use.
Compared with the prior art, the invention has the beneficial effects that:
the Bacillus sp strain has inhibitory activity on various pathogenic bacteria of the tobacco mildew rot, and is determined as Bacillus sp spore bacteria by performing morphological, physiological, biochemical and molecular identification on the strain after determining the activity of the pathogenic bacteria of the tobacco mildew rot through a plurality of batches of activity tests of the pathogenic bacteria of the tobacco mildew rot.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the embodiment of the invention, the preparation and application of the active bacterium Bacillus sp for inhibiting the tobacco mildew rot are as follows: NL-6 bacteria were inoculated into tube LB liquid medium (5 ml per tube) with the following formulation: 0.5% yeast extract, 1% peptone; 1% NaCl. Inoculating NL-6 thalli to a culture medium, and carrying out shake culture at 37 ℃ for 12h at the rotating speed of 220rpm to obtain test tube seeds;
the preparation process of the active bacterium Bacillus sp for inhibiting the tobacco mold rot comprises the steps of transferring test tube seeds into a fresh LB liquid culture medium test tube, and carrying out shake culture at 37 ℃ for 12 hours at the rotating speed of 220rpm to obtain a bacterium solution with strong activity;
100uL of the bacterial liquid is taken to be coated on a fresh LB solid culture medium and cultured in a constant temperature box at 37 ℃ for 12 hours to obtain the Petri dish bacteria.
The preparation process of the active bacterium Bacillus sp for inhibiting the tobacco mildew rot also comprises the following steps of inoculating the test tube strain into a 250ml triangular flask liquid culture medium (100 ml per bottle), wherein the formula of the liquid culture medium is as follows: 0.5% yeast extract; 1% peptone; the remainder of 1% NaCl was water. Culturing at 37 deg.C for 24h with shaking at 220 rpm;
the fermentation culture was centrifuged at 8500rpm/min for 20 minutes, and the supernatant was collected and filtered through a 0.22um filter to obtain a sterile fermentation broth.
Also comprises the following preparation steps:
the method comprises the following steps: the test tube species culture is carried out, and the formula of a culture medium is as follows: 0.5% yeast extract, 1% peptone; 1% NaCl; the rest is water. Inoculating the NL-6 bacillus thalli to a culture medium, and culturing at 37 ℃ for 12h to obtain test tube seeds;
step two: liquid amplification culture, the formula of the liquid culture medium is as follows: 0.5% yeast extract; 1% peptone; 1% NaCl; the rest is water. Inoculating test tube seeds into 500ml of triangular flask liquid culture medium, wherein 200ml of each bottle is filled, and performing shake culture at 37 ℃ for 12h at the rotating speed of 220 rpm;
step three: the formula of the culture medium is as follows: 0.5% yeast extract, 1% peptone; 1% NaCl; 1.5-2% agar. Inoculating the NL-6 bacillus thallus to a culture medium, and culturing at 37 ℃ for 12h to obtain a plate strain.
The method also comprises a test of inhibiting the tobacco mould rotting pathogenic bacteria by the NL-6 bacteria, and specifically comprises the following steps:
the first process is as follows: preparing test bacteria liquid, fungus cakes and the like, inoculating and culturing the NL-6 strain according to the liquid amplification culture method and a petri dish, and preparing the bacteria liquid and the fungus cakes according to the method for inhibiting spore germination and hypha growth of the tobacco mildew and rot pathogenic bacteria.
And preparing a contrast reagent, wherein LB with corresponding amount is adopted as a blank contrast in the test for measuring the inhibition of the NL-6 bacteria on the hypha growth of the tobacco mildew-rot pathogenic bacteria and the test for the spore germination of the fermentation liquor of the NL-6 bacteria on the tobacco mildew-rot pathogenic bacteria.
And thirdly, preparing the tobacco mould-rotting pathogenic bacteria flat plate and the tobacco mould-rotting pathogenic bacteria spores for the test.
The method also comprises the steps of preparing a tobacco mould and rot pathogen bacterium cake, taking down the tobacco mould and rot pathogen bacterium with good growth state from an original plate by using a puncher with the diameter of 1cm, inoculating the tobacco mould and rot pathogen bacterium cake into a new PDA solid plate in a super clean workbench, and culturing the PDA solid plate at the constant temperature of 25 ℃ until the diameter of a bacterial colony is 3cm for later use.
The method further comprises the steps of preparing a spore suspension of the tobacco mildew-rot pathogenic bacteria, inoculating a spore bacterial liquid of the tobacco mildew-rot pathogenic bacteria into a Czochralski liquid culture medium in a 100ml triangular flask according to the proportion of 1:100, and culturing at the constant temperature of 25 ℃ for 180r/min until the spore concentration is 1 × 106-1 × 107cfu/ml for later use.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art through specific situations.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.
Claims (6)
1. The preparation and application of the active bacterium Bacillus sp for inhibiting the tobacco mildew rot are characterized in that the preparation process of the active bacterium Bacillus sp for inhibiting the tobacco mildew rot comprises the following steps: NL-6 bacteria were inoculated into tube LB liquid medium (5 ml per tube) with the following formulation: 0.5% yeast extract, 1% peptone; 1% NaCl. Inoculating NL-6 thalli to a culture medium, and carrying out shake culture at 37 ℃ for 12h at the rotating speed of 220rpm to obtain test tube seeds;
the preparation process of the active bacterium Bacillus sp for inhibiting the tobacco mold rot comprises the steps of transferring test tube seeds into a fresh LB liquid culture medium test tube, and carrying out shake culture at 37 ℃ for 12 hours at the rotating speed of 220rpm to obtain a bacterium solution with strong activity;
100uL of the bacterial liquid is taken to be coated on a fresh LB solid culture medium and cultured in a constant temperature box at 37 ℃ for 12 hours to obtain the Petri dish bacteria.
2. The preparation and application of the active bacterium Bacillus sp for inhibiting the tobacco mildew and rot disease according to claim 1, wherein the preparation process of the active bacterium Bacillus sp for inhibiting the tobacco mildew and rot disease further comprises the following steps of inoculating the test tube strain into a 250ml triangular flask liquid culture medium (100 ml per bottle), wherein the formula of the liquid culture medium is as follows: 0.5% yeast extract; 1% peptone; the remainder of 1% NaCl was water. Culturing at 37 deg.C for 24h with shaking at 220 rpm;
the fermentation culture was centrifuged at 8500rpm/min for 20 minutes, and the supernatant was collected and filtered through a 0.22um filter to obtain a sterile fermentation broth.
3. The preparation and application of the active bacterium Bacillus sp for inhibiting the tobacco mildew rot according to claim 1, characterized by further comprising the following preparation steps:
the method comprises the following steps: the test tube species culture is carried out, and the formula of a culture medium is as follows: 0.5% yeast extract, 1% peptone; 1% NaCl; the rest is water. Inoculating the NL-6 bacillus thalli to a culture medium, and culturing at 37 ℃ for 12h to obtain test tube seeds;
step two: liquid amplification culture, the formula of the liquid culture medium is as follows: 0.5% yeast extract; 1% peptone; 1% NaCl; the rest is water. Inoculating test tube seeds into 500ml of triangular flask liquid culture medium, wherein 200ml of each bottle is filled, and performing shake culture at 37 ℃ for 12h at the rotating speed of 220 rpm;
step three: the formula of the culture medium is as follows: 0.5% yeast extract, 1% peptone; 1% NaCl; 1.5-2% agar. Inoculating the NL-6 bacillus thallus to a culture medium, and culturing at 37 ℃ for 12h to obtain a plate strain.
4. The preparation and application of the active bacterium Bacillus sp for inhibiting the tobacco mildew rot disease according to claim 1, characterized by further comprising a test of NL-6 bacterium for inhibiting the tobacco mildew rot disease source bacterium, and specifically comprising the following procedures:
the first process is as follows: preparing test bacteria liquid, fungus cakes and the like, inoculating and culturing the NL-6 strain according to the liquid amplification culture method and a petri dish, and preparing the bacteria liquid and the fungus cakes according to the method for inhibiting spore germination and hypha growth of the tobacco mildew and rot pathogenic bacteria.
And preparing a contrast reagent, wherein LB with corresponding amount is adopted as a blank contrast in the test for measuring the inhibition of the NL-6 bacteria on the hypha growth of the tobacco mildew-rot pathogenic bacteria and the test for the spore germination of the fermentation liquor of the NL-6 bacteria on the tobacco mildew-rot pathogenic bacteria.
And thirdly, preparing the tobacco mould-rotting pathogenic bacteria flat plate and the tobacco mould-rotting pathogenic bacteria spores for the test.
5. The preparation and application of the active bacterium Bacillus sp for inhibiting the tobacco mildew and rot disease according to claim 1, characterized by further comprising the steps of preparing a cake of the tobacco mildew and rot disease-causing bacterium, taking the tobacco mildew and rot disease-causing bacterium in a good growth state off an original plate by using a hole puncher with the diameter of 1cm, inoculating the bacterium into a new PDA solid plate in a clean bench, and culturing the bacterium at the constant temperature of 25 ℃ until the diameter of a bacterial colony is 3cm for later use.
6. The preparation and application of the active bacterium Bacillus sp for inhibiting the tobacco mildew disease according to claim 1, characterized by further comprising the steps of preparing a spore suspension of the tobacco mildew disease causing bacterium, inoculating a spore bacterial liquid of the tobacco mildew disease causing bacterium into a Czochralski liquid culture medium in a 100ml triangular flask according to a ratio of 1:100, and culturing at a constant temperature of 25 ℃ for 180r/min until the spore concentration is 1 x 106-1 x 107cfu/ml for later use.
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Citations (6)
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| CN102250793A (en) * | 2011-06-20 | 2011-11-23 | 中国农业科学院烟草研究所 | Bacillus pumilus with anti-mildew function |
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2021
- 2021-07-26 CN CN202110842361.0A patent/CN113773979A/en active Pending
Patent Citations (6)
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| CN102250793A (en) * | 2011-06-20 | 2011-11-23 | 中国农业科学院烟草研究所 | Bacillus pumilus with anti-mildew function |
| US20190037851A1 (en) * | 2015-08-28 | 2019-02-07 | Agbiome, Inc | Bacterial strains and their use for controlling plant disease |
| CN107988108A (en) * | 2017-12-20 | 2018-05-04 | 西安交通大学 | A kind of antimycotic bacillus short of money and its antibacterial applications |
| US20200085069A1 (en) * | 2018-09-17 | 2020-03-19 | BiOWiSH Technologies, Inc. | Antifungal composition and method of use |
| CN109136152A (en) * | 2018-09-28 | 2019-01-04 | 四川大学 | One plant for inhibiting production gemma bacillus amyloliquefaciens and its application of plant pathogenic fungi |
| CN112375700A (en) * | 2020-11-11 | 2021-02-19 | 云南省烟草农业科学研究院 | Bacillus belgii 05-1205, acquisition method and application thereof |
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| PAN Z等: "Deciphering the Bacillus amyloliquefaciens B9601-Y2 as a Potential Antagonist of Tobacco Leaf Mildew Pathogen During Flue-Curing", 《FRONT MICROBIOL》 * |
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