CN113881605A - Compound microbial agent for preventing potato scab and preparation method thereof - Google Patents
Compound microbial agent for preventing potato scab and preparation method thereof Download PDFInfo
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- CN113881605A CN113881605A CN202111334788.6A CN202111334788A CN113881605A CN 113881605 A CN113881605 A CN 113881605A CN 202111334788 A CN202111334788 A CN 202111334788A CN 113881605 A CN113881605 A CN 113881605A
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- trichoderma harzianum
- bacillus subtilis
- paecilomyces lilacinus
- powder
- brevibacillus laterosporus
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Classifications
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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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- 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
- A01N63/36—Penicillium
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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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/38—Trichoderma
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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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Abstract
The invention relates to the technical field of agricultural microorganisms, in particular to a compound microbial agent for preventing potato scab, which comprises the following raw materials in parts by weight: 5-25 parts of bacillus subtilis, 5-25 parts of paecilomyces lilacinus, 5-25 parts of trichoderma harzianum, 5-25 parts of bacillus laterosporus and 48-52 parts of glucose. The compound microbial agent disclosed by the invention has strong adaptability and good stability, is beneficial to colonization and activity maintenance of microorganisms at the root of a potato, has a strong antagonistic effect on fungal diseases in the rhizosphere soil of the potato, promotes enrichment of beneficial bacteria at the rhizosphere of the potato, enables the rhizosphere soil of the potato to be changed from a 'fungal type' to a 'bacterial type', reduces the incidence rate of fungal diseases of the potato, can enhance the absorption effect and the fixation effect of the root system of the potato, provides rich nutrient substances for growth of potato plants, and improves the yield and the quality of the potato.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, in particular to a compound microbial agent for preventing potato scab and a preparation method thereof.
Background
The potato is the fourth grain crop next to rice, wheat and corn, the planting area of the potato is wide, the diseases are complex, potato scab is one of the important diseases affecting the production of the potato at present, the potato scab commonly exists in domestic potato production bases, the potato scab mainly damages tubers, brown spots are generated on the surfaces of potato blocks at the initial stage and gradually expand into brown nearly round or indefinite large spots, and the spots are mostly dispersed on the surfaces of the potatoes, so that the appearance of the potatoes is ugly and intolerant to storage, the quality of the potatoes is reduced, the commodity value of the potatoes is reduced, and the potato scab can generally reduce the yield of the potatoes by 10-30%.
At present, comprehensive control measures such as crop rotation, potato seedling and seed disinfection, soil disinfection, cultivation management enhancement, pesticide spraying control and the like are mostly adopted to reduce the incidence rate of potato scab, but the effect is general, a microbial agent is a new direction for preventing the potato scab, the microbial agent can directly or indirectly improve soil, prevent soil-borne diseases and degrade toxic substances, the incidence rate of the potato scab can be effectively reduced, and the product quality of potatoes is improved.
However, most of the current microbial agents for preventing potato scab are applied by irrigation after potatoes are planted, so that the water content in soil is too high, pathogenic bacteria in the soil are easy to propagate in large quantity, the antagonistic action of the microbial agents is reduced, the prevention effect on the potato scab is influenced, and the pathogenic bacteria propagate more seriously especially on the land with a history of the potato scab.
Disclosure of Invention
The first purpose of the invention is to provide a compound microbial agent for preventing potato scab, which has strong adaptability and good stability, is beneficial to colonization and activity maintenance of microorganisms at the root of potatoes, does not need to irrigate the potatoes, improves the antagonistic action of the compound microbial agent, and solves the problem of mass propagation of pathogenic bacteria in soil due to irrigation; the second purpose of the invention is to provide a preparation method of the compound microbial agent for preventing potato scab.
The invention provides a compound microbial agent for preventing potato scab, which comprises the following raw materials in parts by weight: 5-25 parts of bacillus subtilis, 5-25 parts of paecilomyces lilacinus, 5-25 parts of trichoderma harzianum, 5-25 parts of bacillus laterosporus and 48-52 parts of glucose.
The method screens and obtains four strains of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus from the nature, wherein the bacillus subtilis can generate substances similar to cytokinin and plant growth hormone, promotes the growth of potatoes and enables the potatoes to resist the invasion of pathogenic bacteria; the trichoderma harzianum has stronger rhizosphere capability, is easy to colonize at the root of the potato, generates compounds for stimulating the growth of the potato and inducing the defense reaction of the potato, improves the microbial environment of the root system of the potato, enhances the growth vigor and the disease resistance of the potato, and improves the yield and the income of the potato; the paecilomyces lilacinus can inhibit nematode pollution on potato root systems, and indoleacetic acid products generated by metabolism can promote the growth of the potato root systems at low concentration; the brevibacillus laterosporus can enhance photosynthesis, improve the utilization rate of a fertilizer, reduce the content of nitrate, solidify a plurality of heavy metals and reduce the content of the heavy metals in potatoes, the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus act together, glucose is used as a carbon source, the prepared compound microbial inoculum can enhance the absorption and fixation of potato roots, provide rich nutrient substances for the growth of potato plants, can replace partial nitrogenous fertilizer to play the roles of improving soil, improving soil fertility and the like, promote the growth and development of the potato plants, reduce the application amount of the fertilizer, the trichoderma harzianum enables the prepared compound microbial inoculum to be easy to colonize and maintain the activity of the potato roots, the compound microbial inoculum is applied to the positions near the potato roots in a seed dressing and spraying mode without irrigating the potatoes, the condition that a large amount of pathogenic bacteria are propagated due to high water content in soil is avoided; in addition, the bacillus subtilis, the paecilomyces lilacinus and the trichoderma harzianum have synergistic effect, can improve the microbial environment of potato root systems, improve the immunity of potatoes, promote the enrichment of beneficial bacteria in the rhizosphere soil of the potatoes, convert the rhizosphere soil of the potatoes from a fungus type to a bacterium type, reduce the incidence rate of fungal diseases and improve the quality of the potatoes.
Further, the viable count of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus is more than 3 multiplied by 108CFU/g。
Further, the viable count of Bacillus subtilis was 1X 1010CFU/g, viable count of Paecilomyces lilacinus 5 × 109CFU/g, viable count of Trichoderma harzianum 5X 109CFU/g, viable count of Brevibacillus laterosporus is 1 × 1010CFU/g. The bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus obtained by screening are easy to separate and culture, the metabolites are rich, the broad-spectrum antibacterial activity and the stronger stress resistance are achieved, the growth is fast, the stability is good, and the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus can be used as biocontrol bacteria for mass production and application after fermentation treatment.
The invention provides a preparation method of a compound microbial agent for preventing potato scab, which comprises the following steps:
s1, respectively screening and purifying strains of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus, culturing and separating the purified strains to obtain single colonies of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus, and culturing the single colonies again to obtain purified strains of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus;
s2, respectively inoculating purified strains of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus into a culture medium for culturing to obtain liquid seeds of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus;
s3, respectively inoculating liquid seeds of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus to a solid fermentation culture medium for fermentation to obtain strain fermentation liquor of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus;
s4, drying the strain fermentation liquor of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus at constant temperature, and grinding into powder to obtain bacillus subtilis powder, paecilomyces lilacinus powder, trichoderma harzianum powder and brevibacillus laterosporus powder;
s5, uniformly mixing the bacillus subtilis powder, the paecilomyces lilacinus powder, the trichoderma harzianum powder, the brevibacillus laterosporus powder and the glucose to obtain the compound microbial agent.
Further, step S2 is: inoculating the purified strain of the bacillus subtilis to an activation culture medium, and culturing for 46-50h at 35-40 ℃ under the condition of 180-; inoculating the purified strain of the paecilomyces lilacinus to an activation culture medium, and culturing for 46-50h at 26-30 ℃ under the condition of 180-; inoculating the purified strain of trichoderma harzianum to a PDA slant culture medium, culturing at room temperature for 4-6d, and removing conidia of trichoderma harzianum to obtain liquid seeds of trichoderma harzianum; inoculating the purified strain of the Brevibacillus laterosporus into an activation culture medium, and culturing for 46-50h at the temperature of 28-32 ℃ and under the condition of 180-.
Further, in step S3, the solid substrate in the solid fermentation medium includes the following raw materials in parts by weight: 440g/Kg of bran 360-one, 220g/Kg of rice hull powder 180-one, 220g/Kg of corn flour 180-one, 220g/Kg of bean pulp 180-one, 9-11g/Kg of maltose, 18-22g/Kg of tryptone and MgSO413.5-16.5g/Kg。
Further, in step S3, the inoculation amount of the liquid seeds of the bacillus subtilis is 5-10%, the mass ratio of the solid substrate to the water in the solid fermentation culture medium is 1:1, the fermentation temperature is 35-40 ℃, and the fermentation time is 48-72 h;
the inoculation amount of the paecilomyces lilacinus liquid seeds is 5-10%, the mass ratio of the solid substrate to water in the solid fermentation culture medium is 1:1.1, the fermentation temperature is 20-30 ℃, and the fermentation time is 6-9 days.
Further, in step S3, the inoculation amount of the liquid Trichoderma harzianum seeds is 5-10%, the mass ratio of the solid substrate to water in the solid fermentation medium is 1:1.05, the fermentation temperature is 25-28 ℃, and the fermentation time is 13-15 days;
the inoculation amount of the liquid seeds of the brevibacillus laterosporus is 5-10%, the mass ratio of the solid substrate to water in the solid fermentation culture medium is 1:1.1, the fermentation temperature is 25-35 ℃, and the fermentation time is 30-40 h.
According to the method, the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus are respectively subjected to fermentation culture, and the strains can grow in a suitable environment and are not easily polluted by controlling the fermentation temperature and the fermentation time of different strains and the mass ratio of a solid matrix to water in a solid fermentation culture medium, so that a certain amount of high-quality pure strains are obtained, and the compound microbial agent has a more excellent antagonistic effect on fungal diseases.
Further, in step S4, the drying temperature is 38-42 deg.C, and the water content of Bacillus subtilis powder, Paecilomyces lilacinus powder, Trichoderma harzianum powder and Brevibacillus laterosporus powder is less than 10%. When the water content is higher, pathogenic bacteria in the rhizosphere soil of the potatoes can be propagated in a large quantity, and the antagonistic action of the composite microbial agent is reduced.
Further, in step S5, the viable cell count of the composite microbial agent is more than 1X 1010CFU/g. The compound microbial agent prepared by the method is safe and convenient to use, non-toxic, harmless, non-corrosive, harmless to human, animals, plants and non-polluting to the environment, and has ecological safety, and the prepared compound microbial agent is solid powder, low in cost and convenient to transport and use.
The use method of the compound microbial agent comprises the following steps: cutting the germinated potatoes into blocks 1-2 days before sowing, dressing seeds with the compound microbial agent, applying the seed-dressed potatoes into soil, and spraying the compound microbial agent on the grounding parts of the leaves and the potatoes respectively in or after the flowering period.
The invention has the beneficial effects that:
(1) the compound microbial agent has strong adaptability and good stability, is beneficial to colonization and activity maintenance of microorganisms at the root of the potato, has strong antagonistic action on fungal diseases in the rhizosphere soil of the potato, enables the rhizosphere soil of the potato to be changed from a 'fungal type' to a 'bacterial type' by promoting the enrichment of beneficial bacteria in the rhizosphere of the potato, thereby reducing the incidence rate of fungal diseases, inhibiting the occurrence of scab of the potato and improving the quality of the potato.
(2) The bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus are easy to separate and culture, have rich metabolites, broad-spectrum antibacterial activity and strong stress resistance, grow fast and have good stability, and can be used as biocontrol bacteria to be produced and applied in large quantities.
(3) The compound microbial agent is safe and convenient to use, non-toxic and harmless, non-corrosive, harmless to human, animals and plants, non-pollution to the environment and ecological safety, is solid powder, and is low in cost, easy to transport and convenient to use.
(4) The compound microbial agent provided by the invention is used for dressing seeds of potato blocks before sowing, can kill bacteria carried by the potato blocks, avoids external infection, and is sprayed on the grounding parts of the leaves and the potato tubers respectively in or after the flowering phase, so that the compound microbial agent has an antagonistic effect on fungal diseases in soil, and has the effects of increasing yield and improving quality.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. 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.
Example 1
The compound microbial agent for preventing potato scab comprises the following raw materials in parts by weight: 15g of bacillus subtilis, 20g of paecilomyces lilacinus, 7g of trichoderma harzianum, 10g of brevibacillus laterosporus and 48g of glucose.
The preparation method of the compound microbial agent comprises the following steps:
s1, respectively inoculating strains of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus to a solid culture medium for screening and purifying, wherein the viable count of the bacillus subtilis is 1 multiplied by 1010CFU/g, viable count of Paecilomyces lilacinus 5 × 109CFU/g, viable count of Trichoderma harzianum 5X 109CFU/g, viable count of Brevibacillus laterosporus is 1 × 1010CFU/g, culturing and separating the purified strains to obtain single colonies of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus, and culturing the single colonies again to obtain purified strains of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus;
s2, inoculating the purified strain of the bacillus subtilis to an activation culture medium, and culturing for 46h at 35 ℃ and 180r/min to obtain liquid seeds of the bacillus subtilis; inoculating the purified strain of the paecilomyces lilacinus to an activation culture medium, and culturing for 46h at 26 ℃ under the condition of 180r/min to obtain liquid seeds of the paecilomyces lilacinus; inoculating the purified strain of trichoderma harzianum to a PDA slant culture medium, culturing at room temperature for 4d, cleaning to remove conidia of trichoderma harzianum, and obtaining liquid seeds of trichoderma harzianum; inoculating the purified strain of the brevibacillus laterosporus into an activation culture medium, and culturing for 46h under the conditions of 28 ℃ and 180r/min to obtain liquid seeds of the brevibacillus laterosporus;
s3, respectively inoculating liquid seeds of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus into a solid fermentation culture medium for fermentation, wherein the inoculation amount of the liquid seeds of the bacillus subtilis is 8%, the mass ratio of a solid matrix to water in the solid fermentation culture medium is 1:1, the fermentation temperature is 37 ℃, and the fermentation time is 60 hours, so as to obtain strain fermentation liquor of the bacillus subtilis; the inoculation amount of the paecilomyces lilacinus liquid seeds is 10%, the mass ratio of the solid matrix to water in the solid fermentation medium is 1:1.1, the fermentation temperature is 20 ℃, and the fermentation time is 9d, so that the strain fermentation liquor of the paecilomyces lilacinus is obtained; the inoculation amount of the liquid seeds of the trichoderma harzianum is 7 percent, the mass ratio of a solid matrix to water in a solid fermentation culture medium is 1:1.05, the fermentation temperature is 28 ℃, and the fermentation time is 13d, so that strain fermentation liquor of the trichoderma harzianum is obtained; the inoculation amount of the liquid seeds of the brevibacillus laterosporus is 8 percent, the mass ratio of a solid matrix to water in a solid fermentation culture medium is 1:1.1, the fermentation temperature is 30 ℃, and the fermentation time is 35 hours, so that strain fermentation liquor of the brevibacillus laterosporus is obtained;
wherein the solid substrate in the solid fermentation medium comprises the following raw materials in parts by weight: 360g/Kg of bran, 180g/Kg of rice hull powder, 180g/Kg of corn flour, 180g/Kg of soybean meal, 9g/Kg of maltose, 18g/Kg of tryptone and MgSO413.5g/Kg；
S4, drying the strain fermentation liquor of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus at a constant temperature of 38 ℃, and grinding into powder to obtain bacillus subtilis powder, paecilomyces lilacinus powder, trichoderma harzianum powder and brevibacillus laterosporus powder, wherein the water content of the bacillus subtilis powder, the paecilomyces lilacinus powder, the trichoderma harzianum powder and the brevibacillus laterosporus powder is less than 10%;
s5, uniformly mixing the bacillus subtilis powder, the paecilomyces lilacinus powder, the trichoderma harzianum powder, the brevibacillus laterosporus powder and the glucose to obtain the compound microbial agent, wherein the viable count of the compound microbial agent is more than 1 multiplied by 1010CFU/g。
Example 2
The compound microbial agent for preventing potato scab comprises the following raw materials in parts by weight: 5g of bacillus subtilis, 10g of paecilomyces lilacinus, 15g of trichoderma harzianum, 20g of brevibacillus laterosporus and 50g of glucose.
The preparation method of the compound microbial agent comprises the following steps:
s1, respectively inoculating strains of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus to a solid culture medium for screening and purifying, wherein the viable count of the bacillus subtilis is 1 multiplied by 1010CFU/g, viable count of Paecilomyces lilacinus 5 × 109CFU/g, viable count of Trichoderma harzianum 5X 109CFU/g, viable count of Brevibacillus laterosporus is 1 × 1010CFU/g, culturing and separating the purified strains to obtain single colonies of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus, and culturing the single colonies again to obtain purified strains of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus;
s2, inoculating the purified strain of the bacillus subtilis to an activation culture medium, and culturing for 48 hours at the temperature of 38 ℃ and at the speed of 190r/min to obtain liquid seeds of the bacillus subtilis; inoculating the purified strain of the paecilomyces lilacinus to an activation culture medium, and culturing for 48h at the temperature of 28 ℃ and at the speed of 190r/min to obtain liquid seeds of the paecilomyces lilacinus; inoculating the purified strain of trichoderma harzianum to a PDA slant culture medium, culturing at room temperature for 5 days, cleaning to remove conidia of trichoderma harzianum, and obtaining liquid seeds of trichoderma harzianum; inoculating the purified strain of the brevibacillus laterosporus into an activation culture medium, and culturing for 48h under the conditions of 30 ℃ and 190r/min to obtain liquid seeds of the brevibacillus laterosporus;
s3, respectively inoculating liquid seeds of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus into a solid fermentation culture medium for fermentation, wherein the inoculation amount of the bacillus subtilis strain is 7%, the mass ratio of a solid matrix to water in the solid fermentation culture medium is 1:1, the fermentation temperature is 40 ℃, and the fermentation time is 48 hours, so as to obtain a strain fermentation liquid of the bacillus subtilis; the inoculation amount of the paecilomyces lilacinus strain is 7%, the mass ratio of the solid matrix to water in the solid fermentation medium is 1:1.1, the fermentation temperature is 30 ℃, and the fermentation time is 6d, so that the paecilomyces lilacinus strain fermentation liquor is obtained; the inoculation amount of the trichoderma harzianum strain is 8%, the mass ratio of a solid matrix to water in a solid fermentation culture medium is 1:1.05, the fermentation temperature is 27 ℃, and the fermentation time is 14d, so that trichoderma harzianum strain fermentation liquor is obtained; the inoculation amount of the brevibacillus laterosporus strain is 10 percent, the mass ratio of a solid matrix to water in a solid fermentation culture medium is 1:1.1, the fermentation temperature is 25 ℃, and the fermentation time is 40 hours, so that the strain fermentation liquor of the brevibacillus laterosporus is obtained;
wherein the solid substrate in the solid fermentation medium comprises the following raw materials in parts by weight: 400g/Kg of bran, 200g/Kg of rice hull powder, 200g/Kg of corn flour, 200g/Kg of soybean meal, 10g/Kg of maltose, 20g/Kg of tryptone and MgSO4 15g/Kg；
S4, drying the strain fermentation liquor of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus at a constant temperature of 40 ℃, and grinding into powder to obtain bacillus subtilis powder, paecilomyces lilacinus powder, trichoderma harzianum powder and brevibacillus laterosporus powder, wherein the water content of the bacillus subtilis powder, the paecilomyces lilacinus powder, the trichoderma harzianum powder and the brevibacillus laterosporus powder is less than 10%;
s5, mixing the bacillus subtilis powder, the paecilomyces lilacinus powder, the trichoderma harzianum powder, the brevibacillus laterosporus powder and the grapesMixing sugar to obtain compound microbial preparation with viable count of more than 1 × 1010CFU/g。
Example 3
The compound microbial agent for preventing potato scab comprises the following raw materials in parts by weight: 10g of bacillus subtilis, 13g of paecilomyces lilacinus, 20g of trichoderma harzianum, 5g of brevibacillus laterosporus and 52g of glucose.
The preparation method of the compound microbial agent comprises the following steps:
s1, respectively inoculating strains of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus to a solid culture medium for screening and purifying, wherein the viable count of the bacillus subtilis is 1 multiplied by 1010CFU/g, viable count of Paecilomyces lilacinus 5 × 109CFU/g, viable count of Trichoderma harzianum 5X 109CFU/g, viable count of Brevibacillus laterosporus is 1 × 1010CFU/g, culturing and separating the purified strains to obtain single colonies of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus, and culturing the single colonies again to obtain purified strains of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus;
s2, inoculating the purified strain of the bacillus subtilis to an activation culture medium, and culturing for 50h at 40 ℃ at 200r/min to obtain liquid seeds of the bacillus subtilis; inoculating the purified strain of the paecilomyces lilacinus to an activation culture medium, and culturing for 50h at the temperature of 30 ℃ and at the speed of 200r/min to obtain liquid seeds of the paecilomyces lilacinus; inoculating the purified strain of trichoderma harzianum to a PDA slant culture medium, culturing at room temperature for 6 days, cleaning to remove conidia of trichoderma harzianum, and obtaining liquid seeds of trichoderma harzianum; inoculating the purified strain of the brevibacillus laterosporus into an activated culture medium, and culturing for 50h under the conditions of 32 ℃ and 200r/min to obtain liquid seeds of the brevibacillus laterosporus;
s3, respectively inoculating liquid seeds of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus into a solid fermentation culture medium for fermentation, wherein the inoculation amount of the bacillus subtilis strain is 7%, the mass ratio of a solid matrix to water in the solid fermentation culture medium is 1:1, the fermentation temperature is 40 ℃, and the fermentation time is 48 hours, so as to obtain a strain fermentation liquid of the bacillus subtilis; the inoculation amount of the paecilomyces lilacinus strain is 8%, the mass ratio of the solid matrix to water in the solid fermentation medium is 1:1.1, the fermentation temperature is 25 ℃, and the fermentation time is 8d, so that the paecilomyces lilacinus strain fermentation liquor is obtained; the inoculation amount of the trichoderma harzianum strain is 10%, the mass ratio of a solid matrix to water in a solid fermentation culture medium is 1:1.05, the fermentation temperature is 25 ℃, and the fermentation time is 15d, so that trichoderma harzianum strain fermentation liquor is obtained; the inoculation amount of the brevibacillus laterosporus strain is 7 percent, the mass ratio of a solid matrix to water in a solid fermentation culture medium is 1:1.1, the fermentation temperature is 35 ℃, and the fermentation time is 30 hours, so as to obtain the strain fermentation liquor of the brevibacillus laterosporus;
wherein the solid substrate in the solid fermentation medium comprises the following raw materials in parts by weight: 440g/Kg of bran, 220g/Kg of rice hull powder, 220g/Kg of corn flour, 220g/Kg of soybean meal, 11g/Kg of maltose, 22g/Kg of tryptone and MgSO416.5g/Kg；
S4, drying the strain fermentation liquor of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus at a constant temperature of 42 ℃, and grinding into powder to obtain bacillus subtilis powder, paecilomyces lilacinus powder, trichoderma harzianum powder and brevibacillus laterosporus powder, wherein the water content of the bacillus subtilis powder, the paecilomyces lilacinus powder, the trichoderma harzianum powder and the brevibacillus laterosporus powder is less than 10%;
s5, uniformly mixing the bacillus subtilis powder, the paecilomyces lilacinus powder, the trichoderma harzianum powder, the brevibacillus laterosporus powder and the glucose to obtain the compound microbial agent, wherein the viable count of the compound microbial agent is more than 1 multiplied by 1010CFU/g。
Comparative example 1
The raw materials and the preparation method of the compound microbial agent are basically the same as those of the embodiment 2, and the only difference is that the compound microbial agent comprises the following raw materials in parts by weight: 13g of paecilomyces lilacinus, 17g of trichoderma harzianum, 20g of brevibacillus laterosporus and 50g of glucose.
Comparative example 2
The raw materials and the preparation method of the compound microbial agent are basically the same as those of the embodiment 2, and the only difference is that the compound microbial agent comprises the following raw materials in parts by weight: 10g of bacillus subtilis, 15g of paecilomyces lilacinus, 25g of brevibacillus laterosporus and 50g of glucose.
Comparative example 3
The raw materials and the preparation method of the compound microbial agent are basically the same as those in the embodiment 2, and the only difference is that the paecilomyces lilacinus is replaced by the bacillus mucilaginosus.
Comparative example 4
A compound microbial agent has basically the same raw materials and preparation method as example 2, and the only difference is that Bacillus megaterium is used to replace Brevibacillus laterosporus.
Test example
1. Antagonism between strains
Antagonism is a general phenomenon of the microbiology and generally refers to the action of a type of microorganism to inhibit or kill other types of microorganisms. The antagonism experiment is carried out on every two selected strains by adopting the inhibition zone experiment of an agar plate, and the antagonism result is shown in table 1.
TABLE 1
| Bacterial strain | Bacillus subtilis | Paecilomyces lilacinus | Trichoderma harzianum | Brevibacillus laterosporus |
| Bacillus subtilis | - | - | - | - |
| Paecilomyces lilacinus | - | - | - | - |
| Trichoderma harzianum | - | - | - | - |
| Brevibacillus laterosporus | - | - | - | - |
Note: "-" indicates no antagonism.
As can be seen from Table 1, no antagonism exists among Bacillus subtilis, Paecilomyces lilacinus, Trichoderma harzianum and Brevibacillus laterosporus, and strains do not inhibit each other, so that the Bacillus subtilis, Paecilomyces lilacinus, Trichoderma harzianum and Brevibacillus laterosporus can be cooperatively used for preventing crop diseases and insect pests and improving crop yield,
2. evaluation of Effect of Complex microbial Agents
The effect evaluation was performed on the complex microbial agents prepared in examples 1 to 3 and comparative examples 1 to 4, respectively.
The specific test process is as follows:
(1) test soil: the test is carried out in Anyun province of Ganzhou province in Ganzhou region of Zhangye of Ganzhou in 2020, 5-9 months, with soil type of gray irrigated desert soil, moderate fertility, uniform land capability, land length of 5m, width of 4m, and area of 20m2(ii) a The soil basic nutrients to be tested are as follows: 2.4 percent of organic matter, 1.07g/kg of nitrogen and quick-acting phosphorus (P)2O5)9.87mg/kg, quick acting potassium (K)2O)117.48 mg/kg; early crop conditions: potato, 40% infected with scab, test crop potato.
(2) Treatment group:
treatment group 1: conventional fertilization;
treatment group 2: conventional fertilization + the composite microbial inoculum prepared in comparative example 1;
treatment group 3: conventional fertilization + the composite microbial inoculum prepared in comparative example 2;
treatment group 4: conventional fertilization + the composite microbial inoculum prepared in comparative example 3;
treatment group 5: conventional fertilization + the composite microbial inoculum prepared in comparative example 4;
treatment group 6: conventional fertilization + the complex microbial inoculant prepared in example 1;
treatment group 7: conventional fertilization + the complex microbial inoculant prepared in example 2;
treatment group 8: conventional fertilization + complex microbial inoculant prepared in example 3;
each treatment group was set to three replicates and arranged in random blocks, with treatment group 1 being the blank control group.
(3) The operation method comprises the following steps: treatment groups 2-8: 1-2 days before sowing, cutting the germinated potatoes into blocks, respectively adopting the compound microbial agents prepared in examples 1-3 and comparative examples 1-4 to mix seeds on the potato blocks, sowing the potato blocks after seed mixing at 22 days in 5 months, starting thinning and fixing the seedlings when the seedlings are 12-15cm high, enabling the row spacing of the potato plants to be 14cm multiplied by 18cm, carrying out first intertillage, loosening the soil, carrying out second intertillage for 26 days in 6 months, removing weeds, carrying out third intertillage for 13 days in 7 months, ridging, and respectively spraying the compound microbial agents on the grounding parts of blades and potato tubers after the flowering period and the flowering period. The whole growth cycle is totalWatering for three times, applying urea (46%) for 3 times, each time applying urea 75kg/hm2。
Treatment group 1: the conventional fertilization adopts 12000kg/hm of fermented and decomposed cow dung of base fertilizer before sowing2And a commercial compound fertilizer (17-13-14) of 600kg/hm2300kg/hm of diammonium phosphate (18-46-0)2。
The test areas are isolated and separated in a ridging mode, and are respectively treated according to test requirements strictly in the watering process, so that the test areas are not interfered with one another.
(4) Influence of compound microbial agent on potato growing period
The test results are shown in table 2.
TABLE 2
Note: the data in the table are the average of three replicates after sowing.
As can be seen from table 2, the emergence periods of the treatment groups 2 to 8 were advanced by 2d, 3d, 4d, 5d, and the maturation periods were advanced by 6d, 9d, 11d, 15d, and 15d, respectively, as compared with treatment group 1, and the incidence rates of scab of the potatoes of the treatment groups 2 to 8 were significantly reduced, indicating that the composite microbial agents were able to alleviate the occurrence of diseases and effectively reduce the incidence rate of scab prevention, wherein the potatoes of the treatment groups 6 to 8 emerged and matured earliest and none of them were found, indicating that when bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum, and brevibacillus laterosporus, the composite microbial agents had the strongest antagonistic action against the scab pathogenic bacteria of potatoes in the soil and the best effect of scab prevention of potatoes.
(5) Influence of compound microbial inoculant on potato yield
The test results are shown in table 3.
TABLE 3
Note: the data in the table are the average of three replicates after sowing.
As can be seen from Table 3, the emergence rate and the plant height of the treatment groups 2-8 are higher than those of the treatment group 1, the yield of the potatoes is also obviously improved, the yield increase rate is 9.8-44.4%, and the results show that the compound microbial agent can promote the growth of the potato seedlings and improve the yield of the potatoes, wherein the emergence rate, the plant height and the yield increase rate of the potatoes in the treatment groups 6-8 are obviously higher than those of the treatment groups 2-5, and the results show that the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus can stimulate the growth of the potato seedlings more efficiently and less when the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus act synergistically, and the yield increase effect is better.
(6) Influence of compound microbial agent on microorganisms in rhizosphere soil
Respectively taking a soil sample at a position 10cm below the surface soil of the roots of the potato plants in different treatment areas to dilute the soil sample to 10 DEG-6Respectively adding 100 mu L of diluent into a bacterial, fungal and actinomycete culture medium plate, uniformly coating by using a sterile coating rod, standing for 5-10min at room temperature to enable a bacterial liquid to adsorb the culture medium, then putting the culture medium into a thermostat for overnight culture, and calculating the quantity of the bacteria, the fungi and the actinomycete in each gram of soil, wherein the test results are shown in Table 4.
TABLE 4
| Treatment group | Bacterium 105cfu/g | Fungus 103cfu/g | Actinomycetes 105cfu/g |
| 1 | 6.64 | 23.24 | 4.21 |
| 2 | 14.35 | 11.27 | 4.03 |
| 3 | 16.46 | 10.64 | 3.92 |
| 4 | 16.51 | 9.42 | 3.87 |
| 5 | 17.85 | 8.13 | 3.42 |
| 6 | 19.34 | 3.57 | 2.13 |
| 7 | 20.01 | 4.02 | 2.09 |
| 8 | 19.52 | 3.69 | 2.10 |
Note: the data in the table are the average of three replicates after sowing.
As can be seen from Table 4, the number of bacteria in the rhizosphere soil of potatoes in the treatment groups 2 to 8 is higher than that in the treatment group 1, and the number of fungi is lower than that in the treatment group 1, which indicates that the compound microbial agent can increase the number of bacteria in the rhizosphere soil of potatoes, reduce the number of fungi, and convert the rhizosphere soil of potatoes from a fungus type to a bacterium type, so as to reduce the occurrence of fungal diseases, wherein the number of bacteria in the rhizosphere soil of potatoes in the treatment groups 6 to 8 is higher than that in the treatment groups 2 to 5, and the number of fungi is lower than that in the treatment groups 2 to 5, which indicates that when bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus act together, the conversion of fungi can be accelerated, the conversion number of fungi can be increased, and the inhibition effect on the fungal diseases is better.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The compound microbial agent for preventing potato scab is characterized by comprising the following raw materials in parts by weight: 5-25 parts of bacillus subtilis, 5-25 parts of paecilomyces lilacinus, 5-25 parts of trichoderma harzianum, 5-25 parts of bacillus laterosporus and 48-52 parts of glucose.
2. The compound microbial inoculant for preventing potato scab according to claim 1, wherein bacillus subtilis and bacillus pumilus are used as raw materialsThe number of viable bacteria of the paecilomyces purpureus, the trichoderma harzianum and the brevibacillus laterosporus is more than 3 multiplied by 108CFU/g。
3. The compound microbial agent for preventing potato scab according to claim 2, wherein the viable count of bacillus subtilis is 1 x 1010CFU/g, viable count of Paecilomyces lilacinus 5 × 109CFU/g, viable count of Trichoderma harzianum 5X 109CFU/g, viable count of Brevibacillus laterosporus is 1 × 1010CFU/g。
4. A method for preparing the compound microbial inoculant for preventing potato scab as defined in any one of claims 1 to 3, wherein the method comprises the following steps:
s1, respectively screening and purifying strains of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus, culturing and separating the purified strains to obtain single colonies of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus, and culturing the single colonies again to obtain purified strains of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus;
s2, respectively inoculating purified strains of bacillus subtilis, paecilomyces lilacinus, trichoderma harzianum and brevibacillus laterosporus into a culture medium for culturing to obtain liquid seeds of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus;
s3, respectively inoculating liquid seeds of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus to a solid fermentation culture medium for fermentation to obtain strain fermentation liquor of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus;
s4, drying the strain fermentation liquor of the bacillus subtilis, the paecilomyces lilacinus, the trichoderma harzianum and the brevibacillus laterosporus at constant temperature, and grinding into powder to obtain bacillus subtilis powder, paecilomyces lilacinus powder, trichoderma harzianum powder and brevibacillus laterosporus powder;
s5, uniformly mixing the bacillus subtilis powder, the paecilomyces lilacinus powder, the trichoderma harzianum powder, the brevibacillus laterosporus powder and the glucose to obtain the compound microbial agent.
5. The method for preparing a complex microbial inoculant for preventing potato scab according to claim 4, wherein step S2 is: inoculating the purified strain of the bacillus subtilis to an activation culture medium, and culturing for 46-50h at 35-40 ℃ under the condition of 180-; inoculating the purified strain of the paecilomyces lilacinus to an activation culture medium, and culturing for 46-50h at 26-30 ℃ under the condition of 180-; inoculating the purified strain of trichoderma harzianum to a PDA slant culture medium, culturing at room temperature for 4-6d, and removing conidia of trichoderma harzianum to obtain liquid seeds of trichoderma harzianum; inoculating the purified strain of the Brevibacillus laterosporus into an activation culture medium, and culturing for 46-50h at the temperature of 28-32 ℃ and under the condition of 180-.
6. The method for preparing a compound microbial inoculant for preventing potato scab according to claim 4, wherein in step S3, the solid matrix in the solid fermentation medium comprises the following raw materials in parts by weight: 440g/Kg of bran 360-one, 220g/Kg of rice hull powder 180-one, 220g/Kg of corn flour 180-one, 220g/Kg of bean pulp 180-one, 9-11g/Kg of maltose, 18-22g/Kg of tryptone and MgSO413.5-16.5g/Kg。
7. The method for preparing a compound microbial inoculant for preventing potato scab according to claim 6, wherein in step S3, the inoculation amount of liquid seeds of Bacillus subtilis is 5-10%, the mass ratio of solid matrix to water in the solid fermentation medium is 1:1, the fermentation temperature is 35-40 ℃, and the fermentation time is 48-72 h;
the inoculation amount of the paecilomyces lilacinus liquid seeds is 5-10%, the mass ratio of the solid substrate to water in the solid fermentation culture medium is 1:1.1, the fermentation temperature is 20-30 ℃, and the fermentation time is 6-9 days.
8. The method for preparing a compound microbial inoculant for preventing potato scab according to claim 6, wherein in step S3, the inoculation amount of liquid Trichoderma harzianum seeds is 5-10%, the mass ratio of solid matrix to water in the solid fermentation medium is 1:1.05, the fermentation temperature is 25-28 ℃, and the fermentation time is 13-15 days;
the inoculation amount of the liquid seeds of the brevibacillus laterosporus is 5-10%, the mass ratio of the solid substrate to water in the solid fermentation culture medium is 1:1.1, the fermentation temperature is 25-35 ℃, and the fermentation time is 30-40 h.
9. The method for preparing a composite microbial inoculant for preventing potato scab according to claim 4, wherein in step S4, the drying temperature is 38-42 ℃, and the water content of the bacillus subtilis powder, the paecilomyces lilacinus powder, the trichoderma harzianum powder and the brevibacillus laterosporus powder is less than 10%.
10. The method for preparing a complex microbial inoculant for preventing potato scab according to claim 4, wherein in step S5, the number of viable bacteria of the complex microbial inoculant is greater than 1 x 1010CFU/g。
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