CN115039794A - Biocontrol microbial inoculum for preventing and treating tobacco target leaf spot disease as well as preparation method and application thereof - Google Patents
Biocontrol microbial inoculum for preventing and treating tobacco target leaf spot disease as well as preparation method and application thereof Download PDFInfo
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- CN115039794A CN115039794A CN202210758713.9A CN202210758713A CN115039794A CN 115039794 A CN115039794 A CN 115039794A CN 202210758713 A CN202210758713 A CN 202210758713A CN 115039794 A CN115039794 A CN 115039794A
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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
- A01N63/27—Pseudomonas
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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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
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- 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
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- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
- C12R2001/385—Pseudomonas aeruginosa
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Abstract
The invention relates to the field of tobacco disease control methods, and particularly discloses a biocontrol microbial inoculum for controlling tobacco target spots, a preparation method and application thereof, wherein the biocontrol microbial inoculum is formed by mixing pseudomonas aeruginosa bacterial liquid and chitosan, the active ingredient of the pseudomonas aeruginosa bacterial liquid is pseudomonas aeruginosa LK-12, and the preservation number of the pseudomonas aeruginosa LK-12 is CGMCC 10966. The biocontrol microbial inoculum and the diluent thereof can inhibit the growth of rhizoctonia solani, the bacteriostatic effect reaches 85.71 percent, and technical support and theoretical basis are provided for preventing and treating the tobacco target spot disease generated by rhizoctonia solani infection.
Description
Technical Field
The invention relates to the field of tobacco disease control methods, in particular to a biocontrol microbial inoculum for controlling tobacco target spot disease, a preparation method and application thereof.
Background
Tobacco is an annual or limited perennial herb, with the totality of glandular hairs, thick roots, 0.7-2 m high stem and slightly lignified base. The leaves are in the shape of a rectangle circle, a needle, a rectangle circle or an oval, the top end is gradually sharp, the base part is gradually narrowed to the stem to form an ear-shaped half stem, the length is 10-30(-70) cm, the width is 8-15(-30) cm, and the stem is not obvious or becomes a wing-shaped stem. The inflorescence grows at the top, is conical and has a plurality of flowers; the length of the pedicel is 5-20 mm. The calyx is cylindrical or cylindrical bell-shaped, the length is 20-25 mm, and the splinters are triangular and shaped like needles, and have different lengths; the corolla is funnel-shaped and light red, the color of the tube part is lighter and slightly arched, the length of the tube part is 3.5-5 cm, the width of the eave part is 1-1.5 cm, and the splinters are sharp; 1 piece of stamen is obviously shorter than the other 4 pieces, does not extend out of the throat part of the corolla, and the base part of the filament is provided with hair. The shape of the capsule is egg-shaped or rectangular, and the length is approximately equal to the length of the persistent calyx. The seeds are round or wide rectangular round, the diameter is about 0.5 mm, and the seeds are brown. Blossoming and bearing fruit in summer and autumn.
Besides being made into cigarettes, dry tobaccos, pipe tobaccos, cigars and the like for being sucked by people, the tobacco has various medical purposes. Tobacco prefers warm and sunny environment and fertile loose soil, and is drought-resistant and cold-resistant. They are warm and warm in the yang environment, and are not cold-resistant and heat-resistant. Various diseases such as mosaic disease, angular leaf spot and the like are easily generated in the tobacco cultivation process. In recent years, a new disease, namely tobacco target leaf spot, appears in tobacco.
The tobacco target spot disease mainly damages tobacco leaves, is represented as small round water-stain-shaped spots at first, then is rapidly expanded, the disease spots are irregular, the diameter can reach 2-10cm, tissues in the disease spots are light brown and have concentric ring veins, necrotic parts of the disease spots are fragile and fall off to form through holes, and the holes are remained on a target after shooting by bullets, so the disease is called as a target spot disease, and the identification shows that the tobacco target spot disease is mainly caused by the infection of rhizoctonia solani, the rhizoctonia solani belongs to the order of acellular mycoles and the rhizoctonia.
At present, the prevention and control of the tobacco target spot disease are divided into chemical agent prevention and control and biological prevention and control, and the agents recorded in the prior art in the chemical agent prevention and control include azoxystrobin, mancozeb, fluazinam and the like; but long-term use of chemical agents is easy to cause secondary pollution to the environment, and pathogenic bacteria can generate drug resistance; therefore, although biological control is a preferable method, the type of microorganism having a control effect on tobacco target spot disease is single, and the stability of the control effect is not high.
Disclosure of Invention
In order to solve the technical problems, the invention provides a biocontrol microbial inoculum for preventing and treating the tobacco target spot disease, a preparation method and application thereof, wherein the biocontrol microbial inoculum is formed by mixing pseudomonas aeruginosa bacteria liquid and chitosan, the preservation number of the pseudomonas aeruginosa is CGMCC 10966, the biocontrol microbial inoculum can inhibit the growth of rhizoctonia solani, the antibacterial effect reaches 85.71 percent, and technical support and theoretical basis are provided for preventing and treating the tobacco target spot disease generated by the infection of the rhizoctonia solani.
The first purpose of the invention is to provide a biocontrol microbial inoculum for preventing and controlling the target spot disease of tobacco,
the biocontrol microbial inoculum is formed by mixing pseudomonas aeruginosa bacterial liquid and chitosan;
the dosage ratio of the chitosan to the pseudomonas aeruginosa bacterial liquid is 0.8-1 g: 100 mL;
the active ingredient of the pseudomonas aeruginosa bacterial liquid is pseudomonas aeruginosa LK-12;
the preservation number of the pseudomonas aeruginosa LK-12 is CGMCC 10966.
Furthermore, the viable count of the pseudomonas aeruginosa in the biocontrol microbial inoculum is 4-6 x 10 7 cfu/mL。
The second purpose of the invention is to provide a preparation method of the biocontrol microbial inoculum, which comprises the following steps:
s1, preparing a seed solution: performing activation culture on the pseudomonas aeruginosa LK-12 strain under the culture conditions of 32-37 ℃ and 140-;
s2, preparing the biocontrol microbial inoculum: inoculating the seed solution prepared in the S1 into a fresh liquid culture medium according to the inoculation amount of 3-4mL/100mL, culturing at 32-37 ℃ and 140-150r/min for 40-48h to obtain pseudomonas aeruginosa bacterial liquid, and then adding chitosan and mixing uniformly to obtain the biocontrol microbial inoculum.
Further, in S1, the culture conditions of the pseudomonas aeruginosa are 32-37 ℃, 140-150r/min and 20-24 h.
Further, in S1, the culture medium used for activating the pseudomonas aeruginosa is LB solid medium.
Further, in S2, the usage ratio of the chitosan to the pseudomonas aeruginosa bacterial liquid is 0.8-1 g: 100 mL.
Further, in S2, the number of the viable bacteria of the pseudomonas aeruginosa in the biocontrol bacterial agent is 4-6 x 10 7 cfu/mL。
The third purpose of the invention is to provide the application of the biocontrol microbial inoculum in the prevention and treatment of tobacco target spot disease.
Further, the tobacco target spot is caused by rhizoctonia solani infection.
The invention also provides application of the pseudomonas aeruginosa in prevention and treatment of tobacco target spot disease.
Compared with the prior art, the invention has the beneficial effects that:
1. the prepared biocontrol microbial inoculum can inhibit the growth of rhizoctonia solani, the bacteriostatic rate is 85.71%, the biocontrol microbial inoculum prepared by the invention can be used for preventing and treating the tobacco target spot disease caused by the rhizoctonia solani, and technical support and theoretical basis are provided for preventing and treating the tobacco target spot disease;
2. the chitosan in the biocontrol microbial inoculum prepared by the invention can improve the bacteriostatic effect of pseudomonas aeruginosa on rhizoctonia solani.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
The embodiment provides a biocontrol microbial inoculum for preventing and treating target leaf spot of tobacco, a preparation method and application thereof.
Firstly, strain preparation
1. Test strains
(1) Tobacco target leaf blight bacterium (Rhizoctonia solani AG-3) was provided by the plant Virus research laboratory of Shenyang university of agricultural plant protection college;
(2) pseudomonas aeruginosa LK-12 purchased from China general microbiological culture Collection center with the culture collection number of CGMCC 10966.
LB liquid medium: 10g of Tryptone (Tryptone), 5g of Yeast extract (Yeast extract), 10g of sodium chloride (NaCl) and 1000mL of deionized water;
LB solid medium: agar (Agar) is added into the liquid culture medium by 1.5 to 2 percent.
PDA culture medium: 200g of potatoes, 20g of glucose, 15-20 g of agar and 1000ml of distilled water.
2. Preparation of biocontrol microbial inoculum
(1) Activation of Pseudomonas aeruginosa
The purchased pseudomonas aeruginosa LK-12 strain is subjected to activation culture by a streaking method on an LB solid medium flat plate, and the culture conditions are 37 ℃, 150r/min and 24 h. And selecting a single colony on the plate, continuously streaking and transferring for 3-4 times, inoculating the single colony in an LB liquid culture medium, and culturing at 37 ℃ for 24h at 150r/min to obtain a seed solution.
(2) Preparation of biocontrol microbial inoculum
Inoculating the obtained seed solution into a new LB liquid culture medium according to the inoculation amount of 3mL/100mL, culturing at 37 ℃ for 48h at 150r/min to obtain pseudomonas aeruginosa liquid, and then adding chitosan and mixing uniformly to obtain a biocontrol microbial inoculum;
the dosage ratio of the chitosan to the pseudomonas aeruginosa bacterial liquid is 0.9 g: 100 mL.
3. Inhibition effect of biocontrol microbial inoculum on rhizoctonia solani
(1) The plate confronting culture method is adopted to detect the inhibition effect of the biocontrol microbial inoculum on the rhizoctonia solani.
Inoculating rhizoctonia solani on a PDA solid culture medium, culturing to a full culture dish, punching a bacterial cake from the edge of the bacterial colony by using a puncher with the diameter of 5mm, inoculating the bacterial cake at the central position of a fresh blank PDA solid culture medium flat plate, respectively inoculating 2 mu L of biocontrol microbial inoculum at two sides 2.0cm away from the bacterial cake, using sterile water as a control, culturing at a constant temperature of 28 ℃ and observing the bacteriostatic condition, repeating each treatment for three times, measuring the growth radius of the bacterial colony of the pathogenic bacteria when the rhizoctonia solani in the control group PDA solid culture medium flat plate is full of the bacterial strain, and calculating the bacteriostatic rate by using the following formula.
The relative bacteriostasis rate is (control colony radius-treated colony radius)/control colony radius x 100%
(2) Diluting the biocontrol microbial inoculum by 10 times, 30 times, 50 times, 80 times, 100 times, 200 times and 300 times with sterile water, and respectively detecting the relative inhibition rates of the biocontrol microbial inoculum stock solution, the biocontrol microbial inoculum 10 times solution, the biocontrol microbial inoculum 30 times solution, the biocontrol microbial inoculum 50 times solution, the biocontrol microbial inoculum 80 times solution, the biocontrol microbial inoculum 100 times solution, the biocontrol microbial inoculum 200 times solution and the biocontrol microbial inoculum 300 times solution on rhizoctonia solani by the plate confrontation culture method;
each group was run in triplicate and the average was calculated.
Second, experimental results
TABLE 1 bacteriostatic effect of biocontrol microbial inoculum prepared by the invention on rhizoctonia solani
As can be seen from Table 1, the bacteriostat rate of the pseudomonas aeruginosa bacterial liquid to rhizoctonia solani reaches 81.08%, the biocontrol bacterial agent is prepared by adding chitosan, the bacteriostat rate of the biocontrol bacterial agent stock solution to rhizoctonia solani is 85.71%, and the chitosan can improve the bacteriostat effect of the pseudomonas aeruginosa to rhizoctonia solani.
The biocontrol microbial inoculum prepared by the invention can be used for preparing a reagent for inhibiting rhizoctonia solani, and therefore, the biocontrol microbial inoculum prepared by the invention can be used for preventing and treating a tobacco target spot disease caused by the rhizoctonia solani.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. The biocontrol microbial inoculum for preventing and treating the tobacco target spot is characterized in that the biocontrol microbial inoculum is formed by mixing pseudomonas aeruginosa bacterial liquid and chitosan;
the dosage ratio of the chitosan to the pseudomonas aeruginosa bacterial liquid is 0.8-1 g: 100 mL;
the active ingredient of the pseudomonas aeruginosa bacterial liquid is pseudomonas aeruginosa LK-12;
the preservation number of the pseudomonas aeruginosa LK-12 is CGMCC 10966.
2. The biocontrol microbial inoculant according to claim 1 wherein the viable count of pseudomonas aeruginosa in the biocontrol microbial inoculant is between 4 and 6 x 10 7 cfu/mL。
3. The preparation method of the biocontrol microbial inoculum according to claim 1, which is characterized by comprising the following steps:
s1, preparing a seed solution: performing activation culture on the pseudomonas aeruginosa LK-12 strain under the culture conditions of 32-37 ℃ and 140-;
s2, preparing the biocontrol microbial inoculum: inoculating the seed solution prepared in the S1 into a fresh liquid culture medium according to the inoculation amount of 3-4mL/100mL, culturing at 32-37 ℃ and 140-150r/min for 40-48h to obtain pseudomonas aeruginosa bacterial liquid, and then adding chitosan and mixing uniformly to obtain the biocontrol microbial inoculum.
4. The method for preparing the biocontrol microbial inoculum as in claim 3, wherein in S1, the culture conditions of the pseudomonas aeruginosa are 32-37 ℃, 140-150r/min and 20-24 h.
5. The method for preparing a biocontrol microbial inoculum as described in claim 3 wherein in S1, the culture medium used for activating said Pseudomonas aeruginosa is LB solid culture medium.
6. The method for preparing the biocontrol microbial inoculum of claim 3, wherein in S2, the dosage ratio of the chitosan to the pseudomonas aeruginosa bacterial liquid is 0.8-1 g: 100 mL.
7. The method for preparing the biocontrol microbial inoculum of claim 3 wherein in S2 the viable count of Pseudomonas aeruginosa in said biocontrol microbial inoculum is 4-6 x 10 7 cfu/mL。
8. The use of the biocontrol microbial inoculum of claim 1 in the prevention and treatment of tobacco target spot disease.
9. The use of the biocontrol microbial inoculum in the control of tobacco target spot disease according to claim 8, wherein said tobacco target spot disease occurs from rhizoctonia solani dip-staining.
10. Use of pseudomonas aeruginosa according to claim 1 in the control of tobacco target spot disease.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117903950A (en) * | 2023-11-28 | 2024-04-19 | 中国烟草总公司湖南省公司 | Attenuated bacterial strain BY168 for tobacco target spot disease and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117903950A (en) * | 2023-11-28 | 2024-04-19 | 中国烟草总公司湖南省公司 | Attenuated bacterial strain BY168 for tobacco target spot disease and application thereof |
| CN117903950B (en) * | 2023-11-28 | 2024-08-20 | 中国烟草总公司湖南省公司 | Attenuated bacterial strain BY168 for tobacco target spot disease and application thereof |
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