CN113151085A - Compound microbial agent for preventing and treating soil-borne diseases of tobacco and application thereof - Google Patents
Compound microbial agent for preventing and treating soil-borne diseases of tobacco and application thereof Download PDFInfo
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- CN113151085A CN113151085A CN202110443948.4A CN202110443948A CN113151085A CN 113151085 A CN113151085 A CN 113151085A CN 202110443948 A CN202110443948 A CN 202110443948A CN 113151085 A CN113151085 A CN 113151085A
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- tobacco
- paenibacillus polymyxa
- volume fraction
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- bacterial wilt
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- 238000000034 method Methods 0.000 claims abstract description 23
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Images
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C21/00—Methods of fertilising, sowing or planting
-
- 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
-
- 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
- A01N63/25—Paenibacillus
-
- 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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/32—Yeast
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
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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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Abstract
The disclosure relates to the technical field of microbial compound inoculants, and particularly provides a compound microbial inoculants for preventing and treating tobacco soil-borne diseases and application thereof. The Brevibacillus brevis (Brevibacillus brevis) DZQ7 is preserved in China general microbiological culture Collection center in 2018, 4 and 8 months, and the biological preservation number is CGMCC No. 15566. It is combined with Paenibacillus polymyxa YC0136 and Paenibacillus polymyxa YC0573 to prepare a composite microbial inoculum. Solves the problems that the prior microbial inoculum has poor sterilization effect, is not special for the microbial inoculum in tobacco, and can kill germs but often cannot improve the quality of tobacco leaves in the using process of the microbial inoculum.
Description
Technical Field
The disclosure relates to the technical field of microbial compound inoculants, and particularly provides a compound microbial inoculants for preventing and treating tobacco soil-borne diseases and application thereof.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Tobacco is an important economic crop in China and an important source of financial income in China. With the increase of planting years, serious continuous cropping obstacles appear in tobacco fields. The continuous cropping obstacle is characterized by serious soil-borne diseases of tobacco fields, slow growth and development of tobacco and the like. Common soil-borne diseases of tobacco fields include black shank and bacterial wilt. Tobacco black shank is a fungal disease caused by phytophthora nicotianae that has devastating consequences for tobacco production. The tobacco bacterial wilt is a bacterial soil-borne disease caused by ralstonia solanacearum and occurs in most tobacco areas in China, and the tobacco bacterial wilt can cause the reduction or the top-down of tobacco leaves.
At present, a plurality of methods for preventing and treating two diseases exist, including (1) breeding disease-resistant varieties; (2) performing crop rotation with other crops; (3) removing diseased stems and diseased leaves in time, burning or burying the diseased leaves deeply; (4) chemical pesticide control; and (5) biological control. The breeding of disease-resistant varieties has long period, large investment and low efficiency; the effect of preventing and controlling crop rotation with other crops is lower; the stems and leaves of diseases are burnt or buried deeply in time, which causes environmental pollution and increases labor input; the prevention and treatment effect can be enhanced to a certain extent by adopting chemical pesticide, but the drug resistance of pathogenic bacteria can be caused, the production cost is increased, the environmental pollution can be caused, and the safety risk of the tobacco leaves is increased. The biological prevention and control principle has the advantages of short research and development period, low production cost, environmental protection, high efficiency and the like. Therefore, biological control is increasingly gaining attention in the control of tobacco black shank and bacterial wilt.
The microorganisms are important components of biological control, and certain microorganisms can directly inhibit the growth of pathogenic bacteria by secreting secondary metabolites such as antibiotics; some microorganisms can also compete for iron ions with pathogenic bacteria by secreting siderophores so as to indirectly inhibit the growth of the pathogenic bacteria; certain microorganisms can enhance the disease resistance of plants by inducing systemic resistance in the plants.
The inventor finds that the microbial inoculum in the prior art has poor sterilization effect, is not a microbial inoculum specially used in tobacco, and can kill germs but cannot improve the quality of tobacco leaves in the using process.
Disclosure of Invention
Aiming at the problems that the prior microbial inoculum has poor sterilization effect, is not special for the microbial inoculum in tobacco, and can kill germs but often cannot improve the quality of tobacco leaves in the using process of the microbial inoculum.
In one or more embodiments of the disclosure, bacillus brevis (Brevibacillus brevis) DZQ7, which is deposited in the general microbiological center of the China general microbiological culture Collection center at 4-8.2018 and has a biological preservation number of CGMCC No.15566, is provided. And (4) storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
In one or more embodiments of the present disclosure, the application of the brevibacillus brevis DZQ7 in the control of blackleg or bacterial wilt or the synergistic control of blackleg and bacterial wilt is provided.
In one or some embodiments of the present disclosure, there is provided a use of Paenibacillus polymyxa (Paenibacillus polymyxa) YC0136 or Paenibacillus polymyxa (p.polymyxa) YC0573 for the control of blackleg or the synergistic control of blackleg and bacterial wilt.
In one or some embodiments of the present disclosure, a composite microbial agent is provided, which comprises brevibacillus brevis DZQ7, paenibacillus polymyxa YC0136 and paenibacillus polymyxa YC0573 described above.
In one or more embodiments of the present disclosure, an application of the complex microbial agent described above in the control of blackleg or bacterial wilt or the synergistic control of blackleg and bacterial wilt is provided.
In one or some embodiments of the present disclosure, a method for controlling tobacco black shank or bacterial wilt is provided, which comprises the following steps: and after the tobacco is transplanted, irrigating roots by using the compound microbial agent. Diluting the compound microbial agent with clear water, and then irrigating roots of each tobacco plant.
One or some of the above technical solutions have the following advantages or beneficial effects:
1) according to the method, the brevibacillus brevis DZQ7 is screened, and compared with other similar brevibacillus brevis, the brevibacillus brevis DZQ7 has a strong specific antibacterial effect on bacterial wilt or black shank and can be used as a microbial agent.
2) The compound microbial agent disclosed by the invention has a good control effect on tobacco black shank and bacterial wilt, belongs to a biological agent, does not contain chemical insecticides, and does not pollute the environment.
3) The compound microbial agent disclosed by the invention is suitable for application in tobacco, and can promote the agronomic characters of the tobacco while sterilizing. The compound microbial agent can also increase the yield of tobacco leaves, improve the quality of the tobacco leaves and increase the income of tobacco growers to a certain extent.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate embodiments of the disclosure and, together with the description, serve to explain the disclosure and not to limit the disclosure.
FIG. 1 is a plot of the results of the field plot test in example 2, wherein a is control group 3 and b is treatment group.
FIG. 2 is a graph showing the inhibitory effects of Brevibacillus brevis DZQ7, Paenibacillus polymyxa YC0573 and Paenibacillus polymyxa YC0136 in example 1, wherein a is a graph showing the inhibitory effect against Blastomyces nigripes and b is a graph showing the inhibitory effect against Ralstonia solanacearum.
Detailed Description
The technical solutions in the embodiments of the present disclosure 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 disclosure, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the disclosure without making any creative effort, shall fall within the protection scope of the disclosure.
Aiming at the problems that the prior microbial inoculum has poor sterilization effect, is not special for the microbial inoculum in tobacco, and can kill germs but often cannot improve the quality of tobacco leaves in the using process of the microbial inoculum.
In one or more embodiments of the disclosure, a brevibacillus brevis DZQ7 is provided, which has been deposited in the general microbiological center of the China general microbiological culture Collection center at 4-8.2018, and the biological preservation number is CGMCC No. 15566.
In one or more embodiments of the present disclosure, the application of the brevibacillus brevis DZQ7 in the control of blackleg or bacterial wilt or the synergistic control of blackleg and bacterial wilt is provided.
In one or some embodiments of the disclosure, the application of paenibacillus polymyxa YC0136 or paenibacillus polymyxa YC0573 in the control of blackleg or the synergistic control of blackleg and bacterial wilt is provided.
In one or some embodiments of the present disclosure, a composite microbial agent is provided, which comprises brevibacillus brevis DZQ7, paenibacillus polymyxa YC0136 and paenibacillus polymyxa YC0573 described above.
Tests prove that the three bacteria have certain effects on preventing and treating the black shank and the bacterial wilt, but compared with a comprehensive reagent compounded by the three bacteria, the single bacteria has poorer antibacterial performance. And the Brevibacillus brevis DZQ7 disclosed by the disclosure has specificity on black shank and bacterial wilt, and has a better effect compared with similar Brevibacillus brevis.
Preferably, the viable count of the Brevibacillus brevis DZQ7 is more than or equal to 5 x 108The number of viable bacteria of cfu/mL and paenibacillus polymyxa YC0136 is more than or equal to 5 multiplied by 108cfu/mL, the viable count of Paenibacillus polymyxa YC0573 is more than or equal to 5 multiplied by 108cfu/mL；
Preferably, each strain exists in the form of fermentation liquor, wherein the volume fraction of the fermentation liquor of the paenibacillus polymyxa YC0136 is 20-40%, the volume fraction of the fermentation liquor of the paenibacillus polymyxa YC0573 is 20-40%, and the volume fraction of the fermentation liquor of the brevibacillus brevis DZQ7 is 15-30%;
further preferably, the volume fraction of the fermentation liquor of the paenibacillus polymyxa YC0136 is 30%, the volume fraction of the fermentation liquor of the paenibacillus polymyxa YC0573 is 30% and the volume fraction of the fermentation liquor of the brevibacillus brevis DZQ7 is 20%.
Preferably, the feed also comprises saccharomyces cerevisiae and lactobacillus casei;
preferably, the number of viable bacteria of the saccharomyces cerevisiae is more than or equal to 5 multiplied by 108cfu/mL; the viable count of the lactobacillus casei is more than or equal to 5 multiplied by 108cfu/mL；
Preferably, each strain exists in a fermentation liquid form, wherein the volume fraction of the saccharomyces cerevisiae fermentation liquid is 5-15%, and the volume fraction of the lactobacillus casei fermentation liquid is 5-15%;
further preferably, the volume fraction of the saccharomyces cerevisiae fermentation liquor is 10%, and the volume fraction of the lactobacillus casei fermentation liquor is 10%.
Yeast is a unicellular fungus and is not a unit of phylogenetic classification. A micro unicellular microorganism invisible to naked eyes, which can ferment sugar into alcohol and carbon dioxide, is distributed in the whole nature, is a typical heterotrophic facultative anaerobic microorganism, can survive under both aerobic and anaerobic conditions, is a natural leavening agent, and is suitable for being used as cell fermentation liquor.
In one or more embodiments of the present disclosure, there is provided an application of the above complex microbial agent in the control of black shank or bacterial wilt or the synergistic control of black shank and bacterial wilt;
preferably, the compound microbial agent is applied to tobacco.
Experiments prove that the composite microbial inoculum can prevent and treat bacterial wilt and black shank, and has a better effect compared with a single microbial inoculum.
In one or some embodiments of the present disclosure, a method for controlling tobacco black shank or bacterial wilt is provided, which comprises the following steps:
and after the tobacco is transplanted, irrigating roots by using the compound microbial agent. Diluting the compound microbial agent with clear water, and then irrigating roots of each tobacco plant.
The root irrigation fertilization method can play a role in the early stage of tobacco transplantation, has lower requirements on the early stage of tobacco seedlings compared with leaf fertilization and other modes, and can improve the quality of soil while improving the tobacco, namely eliminate phytophthora parasitica and ralstonia solanacearum in the soil.
Preferably, the dilution factor of the clear water is 150-250 times, preferably 200 times;
preferably, the amount of each tobacco root is 150 ml, preferably 200 ml.
Preferably, the compound microbial agent is mixed with a conventional fertilizer (the nutrient content of each mu of land is N: P)2O5:K2O4: 5:10kg) were applied together.
Preferably, the fertilizing amount of the compound microbial inoculum is 2.5-3.5kg per mu of land, and is preferably 3 kg.
Example 1
This example provides the culture methods and activity test results of the strains Paenibacillus polymyxa YC0136, Paenibacillus polymyxa YC0573 and Brevibacillus brevis DZQ 7.
1.1 Strain and tobacco
The strains of the strain comprise Paenibacillus polymyxa YC0136, Paenibacillus polymyxa YC0573 and Brevibacillus brevis DZQ7 which are preserved in China general microbiological culture Collection center with the preservation numbers of CGMCC15562, CGMCC15561 and CGMCC 15566. Saccharomyces cerevisiae and Lactobacillus casei species are commercially available. The tobacco variety used in the field plot experiment was Yunyan 87.
1.2 reagents
Peptone, yeast extract powder, sodium chloride, potato, sucrose, beef extract powder, glucose, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, calcium carbonate, urea, ammonium sulfate, triammonium citrate, sodium acetate, Tween 80, soybean sprout and agar
1.3 Medium
LB liquid medium: 10g of peptone, 5g of yeast extract powder, 10g of sodium chloride, 1000mL of deionized water with constant volume and pH of 7.0.
LB solid medium: 10g of peptone, 5g of yeast extract powder, 10g of sodium chloride, 1000mL of deionized water with constant volume, 7.0 pH and 15g of agar.
PDA liquid culture medium: 200g of potato, 20g of cane sugar and 1000mL of deionized water with a constant volume and natural pH. The preparation method comprises the following steps: peeling potato, cutting into blocks, boiling for half an hour, filtering with gauze, adding sugar, dissolving, adding water to 1000mL, and sterilizing at 121 deg.C for 30 min.
PDA solid medium: 200g of potato, 20g of cane sugar, 1000mL of deionized water with constant volume, natural pH and 15g of agar. The preparation method comprises the following steps: peeling potato, cutting into blocks, boiling for half an hour, filtering with gauze, adding sugar and agar, dissolving, adding water to 1000mL, and sterilizing at 121 deg.C for 30 min.
MRS solid medium: 10g of peptone, 5g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 2g of triammonium citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 801.0 mL of tween, wherein the volume is determined to be 1000mL by deionized water, the pH value is 6.2 +/-0.2, and the agar is 15 g.
Fermentation culture media of paenibacillus polymyxa YC0136 and paenibacillus polymyxa YC 0573: 100g of soybean sprout, 20g of cane sugar, 4g of ammonium sulfate, 1g of urea and 5g of calcium carbonate, and the volume is adjusted to 1000mL by using deionized water, and the pH value is 7.0-7.5. Putting 100g of fresh soybean sprouts into a beaker, adding 1000mL of water, boiling for 30min, and filtering by using gauze. Adding water to the original amount, adding sucrose 20g, ammonium sulfate 4g, urea 1g and calcium carbonate 5g, and sterilizing at 121 deg.C for 20 min.
Fermentation medium of brevibacillus brevis DZQ 7: 100g of soybean sprout, 20g of cane sugar, 4g of ammonium sulfate, 1g of urea and 1g of calcium carbonate, and the volume is adjusted to 1000mL by using deionized water, and the pH value is 7.0-7.5. Putting 100g of fresh soybean sprouts into a beaker, adding 1000mL of water, boiling for 30min, and filtering by using gauze. Adding water to the original amount, adding sucrose 20g, ammonium sulfate 4g, urea 1g and calcium carbonate 1g, and sterilizing at 121 deg.C for 20 min.
Fermentation medium of saccharomyces cerevisiae: 100g of soybean sprouts and 20g of cane sugar are added with deionized water to reach the constant volume of 1000mL, and the pH value is natural. Putting 100g of fresh soybean sprouts into a beaker, adding 1000mL of water, boiling for 30min, and filtering by using gauze. Adding water to the original amount, adding sucrose 20g, and sterilizing at 121 deg.C for 20 min.
Fermentation medium of lactobacillus casei: 100g of soybean sprout, 20g of glucose, 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1g of calcium carbonate, and the pH value of the soybean sprout is 6.2-6.6. Putting 100g of fresh soybean sprouts into a beaker, adding 1000mL of water, boiling for 30min, and filtering by using gauze. Adding water to the original amount, adding 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1g calcium carbonate, pH6.2-6.6, and sterilizing at 121 deg.C for 20 min.
1.4 Strain activation
Selecting Paenibacillus polymyxa YC0136 and Paenibacillus polymyxa YC0573 stored on LB slant culture medium, performing three-zone streaking on the LB culture medium, and culturing at 37 ℃ for 24 h;
selecting the brevibacillus brevis DZQ7 stored on an LB slant culture medium, carrying out three-zone streaking on the LB slant culture medium, and culturing for 24 hours at 37 ℃;
selecting the saccharomyces cerevisiae stored on the PDA slant culture medium, carrying out three-region lineation on the PDA culture medium, and culturing for 24 hours at 28 ℃;
selecting Lactobacillus casei preserved on MRS slant culture medium, performing three-zone streaking on MRS culture medium, and culturing at 37 deg.C for 24 h.
1.5 seed liquid preparation
Selecting a single colony of the activated paenibacillus polymyxa YC0136 strain on an LB solid culture medium, inoculating the single colony into a liquid LB culture medium, culturing at 37 ℃ and 180rpm for 24-48 hours, and taking the single colony as a seed solution;
selecting a single colony of the activated paenibacillus polymyxa YC0573 strain on an LB solid culture medium, inoculating the single colony into a liquid LB culture medium, culturing at 37 ℃ and 180rpm for 24-48 hours, and taking the single colony as a seed solution;
selecting a single colony of the activated brevibacillus brevis DZQ7 strain on an LB solid culture medium, inoculating the single colony into a liquid LB culture medium, culturing at 37 ℃ and 180rpm for 24-48 hours, and taking the single colony as a seed solution;
selecting a single colony of the saccharomyces cerevisiae activated on a PDA solid culture medium, inoculating the single colony into a liquid PDA culture medium, culturing at 28 ℃, 180rpm, and taking the single colony as a seed solution for 24-48 hours;
and (3) selecting a single colony of lactobacillus casei activated on an MRS solid culture medium, inoculating the single colony into a liquid MRS culture medium, and standing and culturing at 37 ℃ for 24-48 hours to serve as seed liquid.
1.6 preparation of fermentation broth
Inoculating seed liquid of cultured Paenibacillus polymyxa YC0136 strain into corresponding fermentation culture medium according to the inoculation amount of 2% -5%, culturing at 37 deg.C and 180rpm, sampling in the fermentation process, and directly adopting microscope techniqueObserving by direct counting method until the viable count of Paenibacillus polymyxa YC0136 is more than or equal to 5 × 108cfu/mL；
Inoculating seed liquid of cultured Paenibacillus polymyxa YC0573 strain into corresponding fermentation culture medium according to the inoculation amount of 2% -5%, culturing at 37 deg.C and 180rpm, sampling in the fermentation process, and observing by direct counting method with microscope direct technique until the viable count of Paenibacillus polymyxa YC0573 is more than or equal to 5 × 108cfu/mL；
Inoculating seed liquid of the cultured brevibacillus brevis DZQ7 strain into corresponding fermentation culture medium according to the inoculation amount of 2% -5%, culturing at 37 ℃ and 180rpm, sampling in the fermentation process, and observing by a direct counting method of a microscope direct technical method until the viable count of the brevibacillus brevis DZQ7 is more than or equal to 5 multiplied by 108 cfu/mL；
Inoculating the seed liquid of the cultured saccharomyces cerevisiae into a corresponding fermentation culture medium according to the inoculation amount of 2-5%, culturing at 28 ℃, and performing 180rpm, sampling in the fermentation process, and observing by a direct counting method of a microscope direct technical method until the viable count of the saccharomyces cerevisiae is more than or equal to 5 multiplied by 108cfu/mL；
Inoculating the seed liquid of cultured lactobacillus casei into corresponding fermentation culture medium according to the inoculation amount of 2% -5%, standing and culturing at 37 ℃, sampling in the fermentation process and observing by a direct counting method of a microscope direct technical method until the viable count of the lactobacillus casei is more than or equal to 5 multiplied by 108cfu/mL。
1.7 bacteriostatic effects of YC0136, YC0573 and DZQ7 plates
Inoculating tobacco black shank bacteria (Phytophthora parasitica var) to PDA solid culture, culturing for 5 days at 28 ℃, taking a mycelium block by using an inoculating hoe, placing the mycelium block in the middle of a solid PDA plate, streaking and inoculating activated brevibacillus brevis DZQ7, paenibacillus polymyxa YC0573 and paenibacillus polymyxa YC0136 at a position about 2cm away from the mycelium block, and culturing for 3 days at 28 ℃ by inverting, as shown in figure 2A. As can be seen from the figure, Brevibacillus brevis DZQ7, Paenibacillus polymyxa YC0573 and Paenibacillus polymyxa YC0136 all show the inhibition effect on the phytophthora parasitica, and the widths of the inhibition zones are 0.6cm, 0.85cm and 0.8cm respectively.
A single colony of Ralstonia solanacearum (Ralstonia solanacearum) which is activated on an LB solid medium is selected and inoculated into a liquid LB medium, the culture is carried out for 36 hours at 37 ℃ and 180rpm, 100 mu L of seed liquid is taken to coat a plate, and the activated DZQ7 is spotted on the plate and is inversely cultured at 37 ℃ overnight. The diameter of the inhibition zone is 1.5-2.6cm, which shows that the Brevibacillus brevis has good inhibition effect on bacterial wilt germs, and is shown in figure 2B. And the paenibacillus polymyxa YC0573 and YC0136 have no obvious inhibition effect on ralstonia solanacearum.
1.8 YC0136, YC0573 and DZQ7 single-bacterium potted plant control effect test on black shank
Uniformly mixing test soil (continuous cropping soil for tobacco planting), removing impurities, keeping the soil content in each pot consistent, culturing in a seedling culture (tobacco variety K326) matrix to 5-6 true leaves, transplanting healthy tobacco seedlings with consistent growth vigor, planting one tobacco seedling in each pot, and burying the exposed stems of the tobacco seedlings into the soil for each treatment of 15 pots. After seedling is released, 108The cfu/mL bacterial solution is diluted in 200mL water and treated by root irrigation, and the corresponding liquid sterile culture medium with the same dilution is used for the control. In order to avoid the influence of illumination and temperature in different directions on the growth of tobacco plants, the flowerpot placement position is randomly adjusted every 10 days, and the management is carried out conventionally. Carrying out hilling after transplanting for 25-35 days, and ensuring that the hilling height of each pot of tobacco is basically kept consistent; the soil humidity is kept between 50 and 60 percent, and the watering is carried out when the maximum water holding capacity is lower than 40 percent, so that the consistent watering amount is ensured.
And (5) investigating the disease condition of the tobacco when the tobacco grows for 60 days and 90 days.
Incidence (%) is ═ 100% (number of diseased tobacco plants/total number of tobacco plants in treatment) ×
The tobacco has black shank disease when being cultured for 60 days. The incidence of the control group was 30%, and the incidence of tobacco black shank was 0 after the use of Brevibacillus brevis DZQ7, Paenibacillus polymyxa YC0573 and Paenibacillus polymyxa YC 0136. The morbidity of the control group at 90 days is 40%, the morbidity of the black shank of the treatment groups of Brevibacillus brevis DZQ7, Paenibacillus polymyxa YC0573 and Paenibacillus polymyxa YC0136 is 20%, 28.6% and 30% respectively, the control groups are obviously superior to the control group, and a certain control effect on the black shank of tobacco is shown.
Example 2
The embodiment provides a compound microbial agent, wherein the compound microbial agent comprises 30% of a paenibacillus polymyxa YC0136 fermentation broth, 30% of a paenibacillus polymyxa YC0573 fermentation broth, 20% of a Brevibacillus brevis DZQ7 fermentation broth, 10% of a saccharomyces cerevisiae fermentation broth and 10% of a lactobacillus casei fermentation broth.
Example 3
Field plot experiment of composite microbial agents
3.1 cell test Point selection
Black shank and bacterial wilt outbreak areas are selected in the Meitan science and technology park in Zunyi Guizhou for testing.
3.2 design of the experiment
Treatment group: 3kg of compound microbial agent per mu of land and conventional fertilization (N: P)2O5:K2The dosage of the O nutrient is 4:5:10 kg); the control group is 3kg Brevibacillus brevis DZQ7+ conventional fertilizer application (N: P) per mu of land2O5:K2The dosage of the O nutrient is 4:5:10 kg). The test design is repeated for 3 times and is distributed randomly, the test area of each cell is 0.1 mu, the planting distance is 1.1m multiplied by 0.6m, and 110 tobacco plants are planted.
3.3 method of use of Complex microbial Agents
And after the tobacco is transplanted, irrigating roots by using the compound microbial agent. The microbial inoculum is diluted by 200 times by clear water, and then the root irrigation is carried out, wherein about 200ml of each strain of tobacco is carried out. It cannot be used together with a bactericidal pesticide. The planting management refers to the GB/T23221-2008 flue-cured tobacco cultivation technical regulation.
3.4 statistics of disease conditions
And (5) investigating the incidence of black shank and bacterial wilt when the tobacco grows for 90 days.
Incidence (%) is (number of diseased tobacco plants per total number of tobacco plants in the plot) × 100%
3.5 agronomic trait statistics
The agronomic characters of the tobacco are investigated when the tobacco grows for 60 days, wherein the agronomic characters comprise plant height, stem circumference, leaf length of the largest leaf and leaf width of the largest leaf, and the index determination refers to a YCT142-1998 tobacco agronomic character investigation method.
3.6 tobacco leaf quality statistics
And (4) baking the harvested tobacco leaves, and then grading the tobacco leaves.
Results
1 composite microbial agent
The composite microbial agent comprises 30 percent of paenibacillus polymyxa YC0136 fermentation broth, 30 percent of paenibacillus polymyxa YC0573 fermentation broth, 20 percent of brevibacillus brevis DZQ7 fermentation broth, 10 percent of saccharomyces cerevisiae fermentation broth and 10 percent of lactobacillus casei fermentation broth.
2 prevention and treatment of tobacco diseases by composite microbial agent under cell condition
2.1 statistics of the control Effect of Black shank
TABLE 1 incidence statistics of black shank
| Treatment of | Incidence (%) |
| Test group | 2.65±1.75 |
| Control group | 8.65±2.95 |
The incidence of tobacco black shank is reduced after the compound microbial inoculum is used. The incidence rate of the black shank in the test group using the compound microbial inoculum is 2.65 percent, and the incidence rate of the black shank in the control group is 8.65 percent. The incidence of blackleg was reduced 69.36% compared to the control.
2.2 statistics of control Effect on bacterial wilt
TABLE 2 statistics of bacterial wilt incidence
| Treatment of | Incidence (%) |
| Test group | 3.25±0.55 |
| Control group | 7.9±1.9 |
The incidence of tobacco bacterial wilt is reduced after the compound microbial agent is used. The incidence of bacterial wilt in the test group using the compound microbial agent is 3.25%, and the incidence of bacterial wilt in the control group is 7.9%. The incidence of blackleg was reduced 58.86% compared to the control.
3 influence of compound microbial agent on agronomic traits of tobacco under plot condition
TABLE 360 day agronomic trait statistics
| Height cm of plant | Circumference of stem | Leaf length cm of maximum leaf | Leaf width cm of maximum leaf | |
| Test of | 87.45±8.65 | 26.8±2.95 | 64.45±6.95 | 31.7±1.7 |
| Control | 83.3±8.1 | 26.725±2.815 | 62.45±5.95 | 28.25±4.15 |
And (5) counting the agronomic characters of the tobacco 60 days after the microbial inoculum is used. The results show that the agronomic characters of the tobaccos of the test groups applied with the compound microbial inoculant are improved to different degrees. Compared with the control, the plant height of the test group is increased by 4.98%, the leaf length of the largest leaf is increased by 3.2%, and the leaf width of the largest leaf is increased by 12.2%.
Influence of compound microbial inoculum on tobacco quality under community condition
TABLE 4 tobacco leaf quality statistics table
Note: the lower orange II X2F, the lower orange III X3F, the middle orange IV C4F and the upper orange III B3F are medium smoke, the middle orange III C3F and the upper orange II B2F are medium smoke, the lower orange IV X4F is low smoke, and the leaves with the mottled color of which the mottled area reaches or exceeds 20 percent are regarded as mottled leaves.
TABLE 5 Effect of Complex microbial Agents on tobacco leaf quality
| Medium and high grade | First class | Lower level | Variegated color | |
| Treatment group | 4.903±0.637 | 3.026±0.695 | 0.109±0.155 | 4.188±1.16 |
| Control group | 4.269±0.196 | 2.349±0.749 | 0.168±0.121 | 3.869±0.752 |
It can be seen from table 5 that the use of the complex microbial inoculant can increase the yield of tobacco leaves. Compared with a control, the yield of the tobacco leaves is increased by 14.74 percent after the compound microbial agent is used. The compound microbial agent can improve the quality of the tobacco leaves while increasing the yield of the tobacco leaves. Compared with the control, the medium tobacco yield of the treatment group is increased by 14.85%, the first tobacco yield is increased by 28.82%, and the lower tobacco yield is decreased by 35.12%.
From the data in tables 1 to 5, the composite microbial inoculum of the embodiment can prevent and treat bacterial wilt and black shank, and has a better effect compared with a single microbial inoculum.
The disclosure of the present invention is not limited to the specific embodiments, but rather to the specific embodiments, the disclosure is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. Brevibacillus brevis (Brevibacillus brevis) DZQ7, which is preserved in China general microbiological culture Collection center in 2018, 4 and 8 months, and the biological preservation number is CGMCC No. 15566.
2. The use of Brevibacillus brevis DZQ7 in preventing and treating Heibustis nigra or bacterial wilt or in preventing and treating Heibustis nigra and bacterial wilt synergistically.
3. Application of paenibacillus polymyxa YC0136 or paenibacillus polymyxa YC0573 in preventing and treating black shank or cooperatively preventing and treating black shank and bacterial wilt.
4. A complex microbial preparation comprising Brevibacillus brevis DZQ7, Paenibacillus polymyxa YC0136 and Paenibacillus polymyxa YC0573 according to claim 1.
5. The complex microbial inoculant according to claim 4, wherein the number of viable bacteria of Brevibacillus brevis DZQ7 is 5 x 10 or more8The number of viable bacteria of cfu/mL and paenibacillus polymyxa YC0136 is more than or equal to 5 multiplied by 108cfu/mL, the viable count of Paenibacillus polymyxa YC0573 is more than or equal to 5 multiplied by 108cfu/mL；
Preferably, each strain exists in the form of fermentation liquor, wherein the volume fraction of the fermentation liquor of the paenibacillus polymyxa YC0136 is 20-40%, the volume fraction of the fermentation liquor of the paenibacillus polymyxa YC0573 is 20-40%, and the volume fraction of the fermentation liquor of the brevibacillus brevis DZQ7 is 15-30%;
further preferably, the volume fraction of the fermentation liquor of the paenibacillus polymyxa YC0136 is 30%, the volume fraction of the fermentation liquor of the paenibacillus polymyxa YC0573 is 30% and the volume fraction of the fermentation liquor of the brevibacillus brevis DZQ7 is 20%.
6. The complex microbial inoculant according to claim 4, further comprising saccharomyces cerevisiae and lactobacillus casei;
preferably, the number of viable bacteria of the saccharomyces cerevisiae is more than or equal to 5 multiplied by 108cfu/mL; the viable count of the lactobacillus casei is more than or equal to 5 multiplied by 108cfu/mL；
Preferably, each strain exists in a fermentation liquid form, wherein the volume fraction of the saccharomyces cerevisiae fermentation liquid is 5-15%, and the volume fraction of the lactobacillus casei fermentation liquid is 5-15%;
further preferably, the volume fraction of the saccharomyces cerevisiae fermentation liquor is 10%, and the volume fraction of the lactobacillus casei fermentation liquor is 10%.
7. The use of the complex microbial inoculant of any one of claims 4-6 for the control of blackleg or bacterial wilt or for the synergistic control of blackleg and bacterial wilt;
preferably, the compound microbial agent is applied to tobacco.
8. A method for preventing and controlling tobacco black shank or bacterial wilt is characterized by comprising the following steps:
after transplanting the tobacco, irrigating roots with the complex microbial inoculant as claimed in any one of claims 4-6. Diluting the compound microbial agent with clear water, and then irrigating roots of each tobacco plant;
preferably, the dilution factor of the clear water is 150-250 times, preferably 200 times;
preferably, the amount of each tobacco root is 150 ml, preferably 200 ml.
9. The method for controlling tobacco black shank or bacterial wilt according to claim 8, wherein the complex microbial agent is applied together with a conventional fertilizer.
10. The method for controlling tobacco black shank or bacterial wilt according to claim 8, wherein the amount of the compound microbial inoculum applied is 2.5-3.5kg, preferably 3kg, per mu of land.
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