CN108373978B - Microbial preparation and application thereof - Google Patents

Microbial preparation and application thereof Download PDF

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CN108373978B
CN108373978B CN201810145996.3A CN201810145996A CN108373978B CN 108373978 B CN108373978 B CN 108373978B CN 201810145996 A CN201810145996 A CN 201810145996A CN 108373978 B CN108373978 B CN 108373978B
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fermentation medium
microbial preparation
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CN108373978A (en
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翟惠佐
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Zibo Vocational Institute
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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
    • C12N1/14—Fungi; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00—Cultivation of specific crops or plants not otherwise provided for
    • A01G22/05—Fruit crops, e.g. strawberries, tomatoes or cucumbers
    • 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
    • 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
    • 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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  • Pest Control & Pesticides (AREA)
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  • Medicinal Chemistry (AREA)
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Abstract

The invention discloses a microbial preparation and application thereof, and belongs to the technical field of microorganisms. The invention adopts photosynthetic bacteria, rhizobium, trichoderma pseudokoningii and other agricultural microorganisms to be compounded with lactobacillus plantarum which can generate synergistic action with growth and metabolism of the agricultural microorganisms to prepare the safe and pollution-free microbial inoculum.

Description

Microbial preparation and application thereof
Technical Field
The invention relates to a microbial preparation and application thereof, belonging to the technical field of microorganisms.
Background
The pepper is a main cultivated plant in China, but the pest and disease damage of the pepper has more and more influence on the yield and the quality of the pepper, and the effective prevention and control of the pest and disease damage of the pepper is the basis of high-quality and high-yield cultivation of the pepper. With the continuous expansion of planting area, pest control is very important for increasing yield and reducing economic loss.
At present, chemical method control is still the main method in the pest control process. Chemical pesticides are widely applied to prevention and control of tomato diseases due to the characteristics of high efficiency, quick effect, convenient operation and the like on the diseases, but excessive use of the pesticides can cause drug resistance of prevention and control objects, destroy soil microbial flora, cause problems of residue, standard exceeding and the like of agricultural products, seriously affect the safety of people and livestock, and even destroy the balance of a natural agricultural ecosystem. With the improvement of the awareness of environmental protection and pollution-free, various methods are explored to reduce the use of chemical pesticides, thereby reducing the harm to people and livestock and environmental pollution. The botanical fungicide as a novel biological agent has the characteristics of no toxicity, no pollution and easy storage and transportation, thereby having wide application range
Disclosure of Invention
The invention aims to solve the first technical problem of providing a microbial preparation for reducing pepper diseases and insect pests.
In one embodiment of the present invention, the microbial preparation is prepared by the following method: rhodopseudomonas, Trichoderma pseudokoningii, rhizobium, Lactobacillus plantarum and Bacillus amyloliquefaciens are respectively subjected to treatment according to the proportion of 1-9.9 multiplied by 105Inoculating the inoculation amount of CFU/mL into the same fermentation medium, and fermenting at 30-35 ℃ for 5-7 days in a sealing way until OD is reached6003.0-3.5, and obtaining the mature cultured microbial inoculum.
In one embodiment of the present invention, the fermentation medium comprises the following components by mass percent: KH (Perkin Elmer)2PO40.05％，K2HPO4·3H2O 0.05％，MgSO4·7H20.05 percent of O, 0.1 percent of NaCl, 1 to 4 percent of urea, 0.2 to 0.5 percent of sodium acetate, 0.01 to 0.06 percent of yeast extract, boiling for 15min, cooling to below 40 ℃, and using NaHCO3Adjusting the pH value to 6.8-7.2.
In one embodiment of the invention, the microbial inoculum is further subjected to scale-up culture, and the scale-up culture comprises the following steps: directly inoculating the mature microbial inoculum into a fermentation medium with the inoculum size of 15-25%, and hermetically fermenting in a transparent container at 30-35 ℃ for 5-7 days until the OD is reached6003.0-3.5, and culturing the microbial inoculum to be mature; the fermentation medium comprises the following components in percentage by mass: KH (Perkin Elmer)2PO40.05％，K2HPO4·3H2O 0.05％，MgSO4·7H20.05 percent of O, 0.1 percent of NaCl, 1 to 4 percent of urea, 0.2 to 0.5 percent of sodium acetate, 0.01 to 0.06 percent of yeast extract, boiling for 15min, cooling to below 40 ℃, and using NaHCO3Adjusting the pH value to 6.8-7.2.
In one embodiment of the invention, the microbial preparation is prepared by placing inoculated bacteria liquid in outdoor sunlight in the culture process, supplementing illumination culture by using an incandescent lamp in cloudy days and at night, wherein the illumination intensity is 1500-2000 lx, and shaking the container for 2-3 times every 8 hours in the culture process to help settled bacteria float upwards to obtain illumination.
The second technical problem to be solved by the invention is to provide a preparation method of a microbial preparation for preventing and treating pepper diseases and insect pests, wherein the method comprises the steps of respectively using rhodopseudomonas, trichoderma pseudokoningii, rhizobium, lactobacillus plantarum and bacillus amyloliquefaciens in a proportion of 1-9.9 multiplied by 105Inoculating the inoculation amount of CFU/mL into the same fermentation medium, placing the same fermentation medium in outdoor sunlight, sealing and fermenting for 5-7 days at the temperature of 30-35 ℃, supplementing illumination culture by using incandescent lamps in cloudy days and at night, wherein the illumination intensity is 1500-2000 lx, and regularly shaking the container for 2-3 times every day in the culture process; to OD6003.0-3.5, and the bacterial liquid is mature.
In one embodiment of the invention, the fermentation medium comprises the following components: KH (Perkin Elmer)2PO40.05％，K2HPO4·3H2O 0.05％，MgSO4·7H20.05 percent of O, 0.1 percent of NaCl, 1 to 4 percent of urea, 0.2 to 0.5 percent of sodium acetate and 0.01 to 0.06 percent of yeast extract.
The third technical problem solved by the invention is to provide the application of the microbial preparation in the production and cultivation of the pepper.
In an embodiment of the invention, the application refers to seed soaking, root dipping and transplanting, root irrigation or liquid level spraying by using microbial inoculum with different dilution times respectively in the pepper cultivation process.
In one embodiment of the invention, the microbial inoculum is diluted by 200-300 times and sprayed for 2-3 days.
The invention has the beneficial effects that: according to the invention, agricultural microorganisms such as photosynthetic bacteria, rhizobia and trichoderma pseudokoningii are compounded with lactobacillus plantarum capable of producing a synergistic effect with growth and metabolism of the agricultural microorganisms to prepare a safe and pollution-free microbial inoculum, so that the emergence rate of pepper seedlings, the seedling height, the overground fresh weight, the main root length and the like can be obviously improved, the product quality and the biological yield of the bud pepper are improved, and the cultivation period is shortened; the composition has obvious effects on preventing and treating powdery mildew, anthracnose, gray mold and aphid, and reduces the incidence rate of the gray mold by 2.32-2.65%, the incidence rate of the anthracnose by 2.3-2.62%, the incidence rate of the aphid by 3.71-4.23% and the incidence rate of the powdery mildew by 2.48-2.83% compared with a control by 2-7%. Meanwhile, the microbial inoculum is simple to prepare, easy to implement, safe and reliable; a shaking table and a large-scale fermentation tank are not needed in the culture process, the fermentation can be carried out in the normal temperature environment in summer, and simple heat preservation measures are only needed in other seasons.
Detailed Description
The method of operation of the present invention is further illustrated below with reference to specific examples. These examples are only for illustrating the present invention in detail and are not intended to limit the scope of the present invention.
Example 1
(1) Preparation of a fermentation medium: the culture medium comprises the following components (by mass percent): KH (Perkin Elmer)2PO40.05％，K2HPO4·3H2O 0.05％，MgSO4·7H20.05 percent of O, 0.1 percent of NaCl, 1 to 4 percent of urea, 0.2 to 0.5 percent of sodium acetate and 0.01 to 0.06 percent of yeast extract, continuously boiling for 15min, and using NaHCO for pH value after cooling3Adjusting the pH value to 6.8-7.2, and subpackaging the culture medium into a sealable transparent fermentation container;
(2) preparation of the microbial inoculum: the microbial inoculum consists of rhodopseudomonas, trichoderma pseudokoningii and rhizobium, and 3 microorganisms are respectively treated according to the proportion of 1-9.9 multiplied by 105Inoculating the strain with the CFU/mL, inoculating the strain in a transparent container, placing the transparent container in outdoor sunlight, sealing and fermenting for 5-7 days at the temperature of 30-35 ℃, supplementing illumination culture by using an incandescent lamp in a cloudy day and at night, maintaining the illumination intensity at 1500-2000 lx, and regularly shaking the container 2-3 times every day in the culture process to help the settled bacteria float upwards to obtain illumination. Cultured to OD600The bacterial liquid color is 2.0-2.5, and the bacterial liquid color is dark red.
(3) Simple amplification culture of the microbial inoculum: in order to conveniently and quickly culture the microbial inoculum in daily production, the bacterial liquid cultured in the step (2) can be inoculated to the culture medium in the step (1) according to the inoculation amount of 20-30%. Fermenting at 30-35 ℃ for 5-7 days in a sealed manner, supplementing illumination culture by using incandescent lamps in cloudy days and at night, maintaining illumination intensity at 1500-2000 lx, and culturing every dayShaking the container 2-3 times regularly to help the settled bacteria float up to obtain light. Cultivating mature bacterial liquid OD600Is 2.0 to 2.5.
In the step (2) and the step (3), the content of viable bacteria in the bacterial liquid can reach 150-200 hundred million/mL.
Example 2
(1) Preparation of a fermentation medium: the culture medium comprises (by mass percent) KH2PO40.05％，K2HPO4·3H2O 0.05％，MgSO4·7H20.05 percent of O, 0.1 percent of NaCl, 1 to 4 percent of urea, 0.2 to 0.5 percent of sodium acetate and 0.01 to 0.06 percent of yeast extract, continuously boiling for 15min, and using NaHCO for pH value after cooling3Adjusting the pH value to 6.8-7.2, and subpackaging the culture medium into a sealable transparent fermentation container;
(2) preparation of the microbial inoculum: the microbial inoculum consists of rhodopseudomonas, trichoderma pseudokoningii, rhizobia, lactobacillus plantarum and bacillus amyloliquefaciens, and 5 microorganisms are respectively treated according to the proportion of 1-9.9 multiplied by 104Inoculating the inoculation amount of CFU/mL into a transparent sealed container, placing the container in outdoor sunlight, sealing and fermenting for 5-7 days at the temperature of 30-35 ℃, supplementing illumination culture by using an incandescent lamp in a cloudy day and at night, maintaining the illumination intensity between 1500-2000 lx, and regularly shaking the container 2-3 times every day in the culture process to help the settled bacteria float upwards to obtain illumination. Cultured to OD6002.7-3.2, and the color of the bacterial liquid is dark red.
(3) Simple amplification culture of the microbial inoculum: in order to conveniently and quickly cultivate the microbial inoculum in daily production, the bacterial liquid cultured in the step (2) can be inoculated in the culture medium in the step (1) according to the inoculation amount of 20-30%, the mixture is sealed and fermented for 5-7 days at 30-35 ℃, an incandescent lamp is adopted to supplement illumination culture in cloudy days and at night, the illumination intensity is maintained between 1500-2000 lx, and the container is periodically shaken for 2-3 times every day in the cultivation process so as to help the precipitated bacteria to float upwards to obtain illumination. Cultivating mature bacterial liquid OD6002.7 to 3.2. In the step (2) and the step (3), the viable bacteria content in the bacterial liquid can reach 100-180 hundred million/mL and 150-200 hundred million/mL respectively.
Example 3
(1) Of fermentation mediaPreparation: the culture medium comprises (by mass percent) KH2PO40.05％，K2HPO4·3H2O 0.05％，MgSO4·7H20.05 percent of O, 0.1 percent of NaCl, 1 to 4 percent of urea, 0.2 to 0.5 percent of sodium acetate and 0.01 to 0.06 percent of yeast extract, continuously boiling for 15min, and using NaHCO for pH value after cooling3Adjusting the pH value to 6.8-7.2, and subpackaging the culture medium into a sealable transparent fermentation container;
(2) preparation of the microbial inoculum: the microbial inoculum consists of rhodopseudomonas, trichoderma pseudokoningii, rhizobia, lactobacillus plantarum and bacillus amyloliquefaciens, and 5 microorganisms are mixed according to the proportion of 1-9.9 multiplied by 105Respectively inoculating the inoculation amounts of CFU/mL into transparent sealed containers, placing in outdoor sunlight, sealing and fermenting at 30-35 ℃ for 5-7 days, supplementing illumination culture with incandescent lamps in cloudy days and at night, maintaining illumination intensity between 1500-2000 lx, and shaking the containers at regular time every day for 2-3 times in the culture process until OD is reached600Is 3.0 to 3.5.
(3) Simple amplification culture of the microbial inoculum: in order to conveniently and quickly cultivate the microbial inoculum in daily production, the bacterial liquid cultured in the step (2) can be inoculated in the culture medium in the step (1) according to the inoculation amount of 20-30%, the mixture is sealed and fermented for 5-7 days at 30-35 ℃, an incandescent lamp is adopted to supplement illumination culture in cloudy days and at night, the illumination intensity is maintained between 1500-2000 lx, the container is periodically shaken for 2-3 times every day in the cultivation process to help the precipitated bacteria to float upwards to obtain illumination culture until OD (optical density) is reached600The bacterial liquid color is 3.0-3.5, and the bacterial liquid color is dark red.
In the step (2) and the step (3), the content of viable bacteria in the bacterial liquid can reach 200-300 hundred million/mL.
Example 4
Respectively diluting the mature culture inocula cultured in the examples 1, 2 and 3 by 200 times, taking a blank as a control, dividing the planting area into A, B, C, D four areas, respectively treating the soaked seeds with the 4 solutions for 2h, then sowing, and covering shallow layers; and after about 20-30 days, irrigating roots of the tomatoes, wherein the root irrigation treatment concentration corresponds to the seed soaking treatment concentration.
On the same day of root irrigation, randomly taking 5 plants from the 4 groups, measuring the height of seedlings, removing clean sandy soil, weighing the fresh weight of each seedling, the fresh weight of the root of each seedling and the fresh weight of the overground part, measuring the length of the main root and the length of the transverse growth development of the root, and calculating the emergence rate of the tomatoes. The results are shown in Table 1.
TABLE 1
Figure BDA0001578854260000041
The results show that the emergence rate of the pepper seedlings treated by the mixed fungicide of the rhodopseudomonas, the trichoderma pseudokoningii, the rhizobia, the lactobacillus plantarum and the bacillus amyloliquefaciens is higher than that of the pepper seedlings treated by the mixed fungicide of the rhodopseudomonas, the trichoderma pseudokoningii and the rhizobium, which indicates that 5 microorganisms can play a synergistic effect and provide various microorganisms, antiviral substances and growth promoting factors for promoting the growth of the pepper seedlings, so that the average seedling height of the cultivated pepper seedlings is 6.31cm higher, and the emergence rate is 32.26% higher than that of the control.
The plant diseases and insect pests in the seedling stage of the pepper are investigated, each area is 20m × 20m, the occurrence degree of the plant diseases and insect pests such as powdery mildew, anthracnose, gray mold, aphid and the like of the pepper treated by the microbial inoculum of different embodiments 3 at different dilution times is recorded, and the results are shown in table 2.
As can be seen from Table 2, the microbial inoculum diluted by 200-300 times has obvious effects on inhibiting powdery mildew, anthracnose, gray mold and aphid, wherein the incidence rate of the gray mold is 2.32-2.65%, the incidence rate of the anthracnose is 2.3-2.62%, the incidence rate of the aphid is 3.71-4.23%, and the incidence rate of the powdery mildew is 2.48-2.83%. Compared with a control, the percentage of the content of the protein is reduced by 2-7 percent.
TABLE 2
Figure BDA0001578854260000051
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (5)

1. The microbial preparation for reducing the pest and disease damage of the pepper is characterized in that the microbial preparation is an active biological product, and the active ingredients of the microbial preparation consist of rhodopseudomonas, trichoderma pseudokoningii, rhizobium, lactobacillus plantarum and bacillus amyloliquefaciens; the preparation method comprises the following steps: rhodopseudomonas, Trichoderma pseudokoningii, rhizobium, Lactobacillus plantarum and Bacillus amyloliquefaciens are respectively subjected to treatment according to the proportion of 1-9.9 multiplied by 105Inoculating the inoculation amount of CFU/mL into the same fermentation medium, placing the same fermentation medium in outdoor sunlight, sealing and fermenting for 5-7 days at the temperature of 30-35 ℃, supplementing illumination culture by using incandescent lamps in cloudy days and at night, wherein the illumination intensity is 1500-2000 lx, and regularly shaking the container for 2-3 times every day in the culture process; up to OD6003.0-3.5, and obtaining a mature cultured microbial inoculum; the fermentation medium comprises the following components: KH (Perkin Elmer)2PO4 0.05%，K2HPO4·3H2O 0.05%，MgSO4·7H20.05% of O, 0.1% of NaCl, 1-4% of urea, 0.2-0.5% of sodium acetate and 0.01-0.06% of yeast extract.
2. The microbial preparation according to claim 1, wherein the fermentation medium comprises the following components in percentage by mass: KH (Perkin Elmer)2PO4 0.05%，K2HPO4·3H2O 0.05%，MgSO4·7H20.05% of O, 0.1% of NaCl, 1-4% of urea, 0.2-0.5% of sodium acetate, 0.01-0.06% of yeast extract, boiling for 15min, cooling to below 40 ℃, and using NaHCO3Adjusting the pH value to 6.8-7.2.
3. The microbial preparation of claim 1, wherein the microbial preparation is further subjected to an expansion culture, wherein the expansion culture comprises the following steps: directly inoculating the mature microbial inoculum into a fermentation medium with the inoculum size of 15-25%, and hermetically fermenting in a transparent container at 30-35 ℃ for 5-7 days until the OD is reached6003.0-3.5, and culturing the microbial inoculum to be mature; the fermentation medium comprises the following components in percentage by mass: KH (Perkin Elmer)2PO4 0.05%，K2HPO4·3H2O 0.05%，MgSO4·7H20.05% of O, 0.1% of NaCl, 1-4% of urea, 0.2-0.5% of sodium acetate, 0.01-0.06% of yeast extract, boiling for 15min, cooling to below 40 ℃, and using NaHCO3Adjusting the pH value to 6.8-7.2.
4. The microbial preparation according to claim 1, wherein the inoculated bacterial solution is placed in the outdoor sunlight in the culture process, an incandescent lamp is adopted to supplement illumination culture in cloudy days and at night, the illumination intensity is 1500-2000 lx, and the container is shaken for 2-3 times every 8 hours in the culture process to help the settled bacteria float upwards to obtain illumination.
5. A process for preparing the microbial preparation of claim 1, wherein the process comprises treating Rhodopseudomonas sp, Trichoderma pseudokoningii, Rhizobium sp, Lactobacillus plantarum, and Bacillus amyloliquefaciens with the ratio of 1-9.9X 105Inoculating the inoculation amount of CFU/mL into the same fermentation medium, placing the same fermentation medium in outdoor sunlight, sealing and fermenting for 5-7 days at the temperature of 30-35 ℃, supplementing illumination culture by using incandescent lamps in cloudy days and at night, wherein the illumination intensity is 1500-2000 lx, and regularly shaking the container for 2-3 times every day in the culture process; to the bacterial liquid OD6003.0-3.5, and cultivating the bacterial liquid to be mature; the fermentation medium comprises the following components: KH (Perkin Elmer)2PO4 0.05%，K2HPO4·3H2O 0.05%，MgSO4·7H20.05% of O, 0.1% of NaCl, 1-4% of urea, 0.2-0.5% of sodium acetate and 0.01-0.06% of yeast extract.
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