CN117821331A - Composite microbial agent and preparation method and application thereof - Google Patents
Composite microbial agent and preparation method and application thereof Download PDFInfo
- Publication number
- CN117821331A CN117821331A CN202410038813.3A CN202410038813A CN117821331A CN 117821331 A CN117821331 A CN 117821331A CN 202410038813 A CN202410038813 A CN 202410038813A CN 117821331 A CN117821331 A CN 117821331A
- Authority
- CN
- China
- Prior art keywords
- bacterial
- bacillus
- liquid
- microbial agent
- mixed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Plant Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pest Control & Pesticides (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Dentistry (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Agronomy & Crop Science (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a composite microbial agent, a preparation method and application thereof, and belongs to the technical field of microorganisms. The microbial inoculum comprises mixed bacterial liquid and a bacterial accelerator. The solid-liquid ratio of the mixed bacterial liquid and the bacterial seed accelerant is 100mL:5-10g. The mixed bacterial liquid consists of bacillus bailii bacterial suspension, bacillus aryabhattai bacterial suspension and bacillus circulans bacterial suspension, and the volume ratio of the three bacterial suspensions is 1:1:1. The method for preparing the composite microbial agent by screening three functional microbial strains has the advantages of obvious prevention and control effects on potato scab due to the synergistic effect of the three functional microbial strains, capability of greatly improving the yield and quality of potatoes, no toxicity and harm, convenience in use and no environmental pollution, and has a good application prospect in the field of agricultural planting.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a composite microbial agent, a preparation method and application thereof.
Background
Potato (Solanum tuberosum l.) is one of the world's important crops, is widely planted and used in food processing for human consumption, and has become one of the four main grain crops in our country. The potato scab at the present stage is the fourth disease of the potato, mainly the soil transmission, and the situation of seed potato disease frequently occurs. The potato epidermis after scab infection can appear as concave or convex lesions, become rough, have poor appearance phenotype, remarkably reduce quality, lose commodity and market value, and are very destructive to the potato industry. However, with the continuous improvement of the state and even international status of potatoes and the important role of protecting the national food safety, the research on potato scab is not slow.
Potato scab is a soil-borne and seed-borne disease caused by streptomyces scab of the order actinomycetes. Scab has been regarded as a worldwide problem of soil-borne diseases and has been paid attention to by various nations. The control direction of potato scab at the present stage is mainly disease-resistant variety breeding, and the control by means of chemistry, biology, agriculture and the like is also a research direction.
The chemical agent is widely used in field production, and is the most convenient method for solving plant diseases. However, the problems of pesticide residue, environmental pollution and the like are the pain spot problem caused by using chemical agents, and particularly the chemical agents can also generate drug resistance after long-term use, so the chemical agents are not the optimal choice for solving potato scab.
Agricultural control is an agricultural comprehensive technical measure which can enhance the resistance of crops to diseases by improving and regulating the growth environment of crops. Potato scab is a very serious soil-borne disease that can reduce the spread of pathogenic bacteria by means of soil rotation. However, the agricultural control means has slow effect, long period and poor stability, and can only be used as an auxiliary means for actual agricultural production.
With the continuous development of agriculture, biological control of diseases is increasingly gaining importance. The probiotics can participate in the defending function of plants, enhance the stress resistance, virus resistance and animal hazard resistance of the plants, and can also provide nitrogen sources, hormones and other nutrient substances for the plants. Plant probiotics are biological control agents, and their metabolites can control viruses, bacteria and fungi, promote the absorption of N, P, K by plants, and can prevent abiotic injury and the like. However, the existing microbial control means have no pertinence, single functions and unstable effects, so that effective application and popularization are difficult to obtain.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the compound microbial agent for effectively preventing and controlling the potato scab, and the selected biological bacterial strain can not only efficiently antagonize pathogenic bacteria of the potato scab, but also stimulate crop growth, and improve the yield and quality of the potato.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a composite microbial agent comprises mixed bacterial liquid and strain promoter.
Preferably, the solid-to-liquid ratio of the mixed bacterial liquid and the bacterial seed accelerator is 100mL:5-10g.
Preferably, the mixed bacterial liquid comprises bacillus bailii (Bacillus velezensis) bacterial suspension, bacillus aryabhattai (Bacillus aryabhattai) bacterial suspension and bacillus circulans (Bacillus circulans) bacterial suspension, and the volume ratio of the three bacterial suspensions is 1:1:1.
More preferably, the bacillus belicus is purchased from China general microbiological culture collection center (CGMCC) with a collection number of CGMCC No.1.12669, and the original collection time is as follows: 2013, 9 and 10; the bacillus arvensis is purchased from China general microbiological culture collection center (CGMCC) with a collection number of 1.16121 and an original collection time of 2019, 3 months and 20 days; the bacillus circulans is purchased from China general microbiological culture collection center (CGMCC) with a collection number of CGMCC No.1.8819 and an original collection time of 2008, 10 months and 21 days. All three strains used in the invention can be inquired in the strain catalogue of China general microbiological culture collection center, and open purchase is provided without repeated preservation.
Preferably, the strain promoter comprises glucose, whey powder, ascorbic acid and ammonium molybdate, and the mass ratio of the strain promoter is (40-50)/(10-20)/(2-5)/(1-3).
The preparation method of the composite microbial agent comprises the following preparation steps:
A. activating Bacillus bailii, bacillus aryabhattai and Bacillus circulans in LB solid culture medium, respectively, inoculating single colony into test tube of LB liquid culture medium, and culturing at 28-30deg.C and 180-200r/min to OD 600 =0.8, resulting in seed solution; inoculating the seed solution into 200mLLB liquid culture medium according to the amount of 1%, culturing for 12h at 28-30 ℃ at 180-200r/min, washing the seed solution with sterile PBS buffer solution for 3 times, and then re-suspending the seed solution to obtain three bacterial suspensions, and mixing the three bacterial suspensions according to the volume ratio of 1:1:1 to obtain mixed bacterial solution;
B. glucose, whey powder, ascorbic acid and ammonium molybdate are prepared according to a certain proportion, and are uniformly mixed to form a strain promoter;
C. packaging the mixed bacterial liquid and the bacterial accelerant respectively, wherein when the mixed bacterial liquid and the bacterial accelerant are used, the solid-liquid ratio is 100mL:5-10g, diluted 500-800 times and applied.
More preferably, the preparation method of the LB liquid medium in the step A comprises the following steps: mixing 1L of water, 10g of tryptone, 5g of yeast extract and 10g of NaCl until solute is dissolved, and regulating the pH to 7.0 by using 3-5mol/L of NaOH to obtain an LB liquid culture medium; the LB solid culture medium is obtained by adding 15-20g agar based on LB liquid culture medium.
More preferably, the effective viable count in the mixed bacterial liquid obtained in the step A is not less than 1 multiplied by 10 9 CFU/mL。
An application of a compound microbial agent for preventing and treating potato scab.
The chemical agent has rapid and efficient control effect on scab, but the soil is treated by the chemical agent to influence soil organisms, particularly the beneficial microorganism community is greatly negatively influenced, and the irreversible 'directional screening' effect is realized on the soil microorganism population, so that the potato scab is aggravated when the chemical agent is used. Therefore, changing the soil microenvironment and increasing the enrichment of beneficial bacteria on the tuber surface is the best method for preventing and controlling potato scab.
The invention uses three bacillus biocontrol bactericides to form functional microorganisms, the bacillus biocontrol bactericides have wide physiological functions, can generate endospores, have certain resistance to severe environment, can stably exist in plant rhizosphere and soil, secrete various bioactive substances, and have the advantages of broad-spectrum bacteriostasis and the like.
The invention screens and compounds three biocontrol function microorganism strains to form a composite microbial agent, wherein the selected bacillus belicus has broad-spectrum antibacterial property, can secrete antibacterial substances such as LCI protein and the like, and has good inhibition effect on pathogenic bacteria such as streptomyces scab and the like; the selected bacillus aryabhattai can secrete the metabolite to inhibit the growth of streptomyces scab mycelium, reduce the number of pathogenic bacteria in soil and inhibit the incidence of scab; the bacillus circulans not only has broad-spectrum bacteriostasis, but also can produce various metabolites, induce plants to produce resistance to regulate over-expression of defensive enzyme genes, improve the defensive capacity of the plants, secrete plant hormones such as auxin and the like, promote plant growth and improve the yield and quality of crops. After the three are mixed in equal proportion, effective synergy is generated, the effective control of potato scab is realized, the growth of potatoes is promoted, and the yield and quality of the potatoes are improved.
The invention also compounds glucose, whey powder, ascorbic acid, ammonium molybdate and other bacterial promoters to provide nutrients for the bacterial agents and stimulate the activity of the bacterial strains, so that the functional bacterial strains can efficiently play roles.
Advantageous effects
The method for preparing the composite microbial agent by screening three functional microbial strains has remarkable prevention and control effects on potato scab, greatly improves potato yield and quality, is nontoxic and harmless, is convenient to use, does not pollute the environment, and has good application prospects in the field of agricultural planting.
Drawings
FIG. 1 is a graph showing the inhibitory effects of three strains of the present invention on Streptomyces scab, wherein a is Bacillus belicus, b is Bacillus aryabhattai, and c is Bacillus circulans.
Detailed Description
The technical scheme of the present invention is further described below with reference to specific examples, but is not limited thereto.
Example 1
A composite microbial agent comprises mixed bacterial liquid and strain promoter.
The solid-liquid ratio of the mixed bacterial liquid and the bacterial seed accelerant is 100mL:5g.
The mixed bacterial liquid consists of bacillus bailii (Bacillus velezensis) bacterial suspension, bacillus aryabhattai (Bacillus aryabhattai) bacterial suspension and bacillus circulans (Bacillus circulans) bacterial suspension, and the volume ratio of the three bacterial suspensions is 1:1:1.
The bacillus belicus is purchased from China general microbiological culture collection center (CGMCC) with a collection number of CGMCC No.1.12669, and the original collection time is as follows: 2013, 9 and 10; the bacillus arvensis is purchased from China general microbiological culture collection center (CGMCC) with a collection number of 1.16121 and an original collection time of 2019, 3 months and 20 days; the bacillus circulans is purchased from China general microbiological culture collection center (CGMCC) No.1.8819, and the original preservation time is 10 months and 21 days in 2008. All three strains used in the embodiment can be inquired through the strain catalogue of China general microbiological culture Collection center, and open purchase is provided without repeated preservation.
The strain promoter comprises glucose, whey powder, ascorbic acid and ammonium molybdate with a mass ratio of 40:10:2:1.
The preparation method of the composite microbial agent comprises the following preparation steps:
A. activating Bacillus bailii, bacillus albe and Bacillus circulans in LB solid medium, respectively, picking single colony, inoculating into test tube of LB liquid medium, and culturing at 28-30deg.C and 180r/min to OD 600 =0.8, resulting in seed solution; inoculating the seed solution into 200mLLB liquid culture medium according to the amount of 1%, culturing for 12h at 28-30 ℃ at 180r/min, washing the seed solution for 3 times by using sterile PBS buffer solution, and then re-suspending the seed solution to obtain three bacterial suspensions, and mixing the three bacterial suspensions according to the volume ratio of 1:1:1 to obtain mixed bacterial solution;
B. glucose, whey powder, ascorbic acid and ammonium molybdate are prepared according to a certain proportion, and are uniformly mixed to form a strain promoter;
C. packaging the mixed bacterial liquid and the bacterial accelerant respectively, wherein when the mixed bacterial liquid and the bacterial accelerant are used, the solid-liquid ratio is 100mL:5g, diluted 500-800 times and applied.
The preparation method of the LB liquid medium in the step A comprises the following steps: mixing 1L of water, 10g of tryptone, 5g of yeast extract and 10g of NaCl uniformly until solute is dissolved, and regulating the pH to 7.0 by using 3mol/L NaOH to obtain an LB liquid culture medium; the LB solid culture medium is obtained by adding 15g of agar on the basis of LB liquid culture medium.
The effective viable count in the mixed bacterial liquid obtained in the step A is not less than 1 multiplied by 10 9 CFU/mL。
An application of a compound microbial agent for preventing and treating potato scab.
Example 2
A composite microbial agent comprises mixed bacterial liquid and strain promoter.
The solid-liquid ratio of the mixed bacterial liquid and the bacterial seed accelerant is 100mL:8g
The mixed bacterial liquid consists of bacillus bailii (Bacillus velezensis) bacterial suspension, bacillus aryabhattai (Bacillus aryabhattai) bacterial suspension and bacillus circulans (Bacillus circulans) bacterial suspension, and the volume ratio of the three bacterial suspensions is 1:1:1.
The bacillus belicus is purchased from China general microbiological culture collection center (CGMCC) with a collection number of CGMCC No.1.12669, and the original collection time is as follows: 2013, 9 and 10; the bacillus arvensis is purchased from China general microbiological culture collection center (CGMCC) with a collection number of 1.16121 and an original collection time of 2019, 3 months and 20 days; the bacillus circulans is purchased from China general microbiological culture collection center (CGMCC) No.1.8819, and the original preservation time is 10 months and 21 days in 2008. All three strains used in the embodiment can be inquired through the strain catalogue of China general microbiological culture Collection center, and open purchase is provided without repeated preservation.
The strain promoter comprises glucose, whey powder, ascorbic acid and ammonium molybdate, and the mass ratio is 45:15:3:2.
The preparation method of the composite microbial agent comprises the following preparation steps:
A. activating Bacillus bailii, bacillus albe and Bacillus circulans in LB solid medium, respectively, picking single colony, inoculating into test tube of LB liquid medium, and culturing at 28-30deg.C and 180r/min to OD 600 =0.8, resulting in seed solution; inoculating the seed solution into 200mLLB liquid culture medium according to the amount of 1%, culturing for 12h at 28-30 ℃ at 180r/min, washing the seed solution for 3 times by using sterile PBS buffer solution, and then re-suspending the seed solution to obtain three bacterial suspensions, and mixing the three bacterial suspensions according to the volume ratio of 1:1:1 to obtain mixed bacterial solution;
B. glucose, whey powder, ascorbic acid and ammonium molybdate are prepared according to a certain proportion, and are uniformly mixed to form a strain promoter;
C. packaging the mixed bacterial liquid and the bacterial accelerant respectively, wherein when the mixed bacterial liquid and the bacterial accelerant are used, the solid-liquid ratio is 100mL:8g, diluted 500-800 times and applied.
The preparation method of the LB liquid medium in the step A comprises the following steps: mixing 1L of water, 10g of tryptone, 5g of yeast extract and 10g of NaCl uniformly until solute is dissolved, and regulating the pH to 7.0 by using 3mol/L NaOH to obtain an LB liquid culture medium; the LB solid culture medium is obtained by adding 15g of agar on the basis of LB liquid culture medium.
The effective viable count in the mixed bacterial liquid obtained in the step A is not less than 1 multiplied by 10 9 CFU/mL。
An application of a compound microbial agent for preventing and treating potato scab.
Example 3
A composite microbial agent comprises mixed bacterial liquid and strain promoter.
The solid-liquid ratio of the mixed bacterial liquid and the bacterial seed accelerant is 100mL:10g.
The mixed bacterial liquid consists of bacillus bailii (Bacillus velezensis) bacterial suspension, bacillus aryabhattai (Bacillus aryabhattai) bacterial suspension and bacillus circulans (Bacillus circulans) bacterial suspension, and the volume ratio of the three bacterial suspensions is 1:1:1.
The bacillus belicus is purchased from China general microbiological culture collection center (CGMCC) with a collection number of CGMCC No.1.12669, and the original collection time is as follows: 2013, 9 and 10; the bacillus arvensis is purchased from China general microbiological culture collection center (CGMCC) with a collection number of 1.16121 and an original collection time of 2019, 3 months and 20 days; the bacillus circulans is purchased from China general microbiological culture collection center (CGMCC) No.1.8819, and the original preservation time is 10 months and 21 days in 2008. All three strains used in the embodiment can be inquired through the strain catalogue of China general microbiological culture Collection center, and open purchase is provided without repeated preservation.
The strain promoter comprises glucose, whey powder, ascorbic acid and ammonium molybdate in a mass ratio of 50:20:5:3.
The preparation method of the composite microbial agent comprises the following preparation steps:
A. activating Bacillus bailii, bacillus aryabhattai and Bacillus circulans in LB solid culture medium, respectively, picking single colony, inoculating into test tube of LB liquid culture medium, and culturing at 28-30deg.C and 200r/min to OD 600 =0.8, getTo seed liquid; inoculating the seed solution into 200mLLB liquid culture medium according to the amount of 1%, culturing for 12h at 28-30 ℃ at 200r/min, washing the seed solution for 3 times by using sterile PBS buffer solution, and then re-suspending the seed solution to obtain three bacterial suspensions, and mixing the three bacterial suspensions according to the volume ratio of 1:1:1 to obtain mixed bacterial solution;
B. glucose, whey powder, ascorbic acid and ammonium molybdate are prepared according to a certain proportion, and are uniformly mixed to form a strain promoter;
C. packaging the mixed bacterial liquid and the bacterial accelerant respectively, wherein when the mixed bacterial liquid and the bacterial accelerant are used, the solid-liquid ratio is 100mL:10g of the mixture is diluted 500-800 times and then applied.
The preparation method of the LB liquid medium in the step A comprises the following steps: mixing 1L of water, 10g of tryptone, 5g of yeast extract and 10g of NaCl until solute is dissolved, and regulating the pH to 7.0 by using 3-5mol/L of NaOH to obtain an LB liquid culture medium; the LB solid culture medium is obtained by adding 20g of agar based on LB liquid culture medium.
The effective viable count in the mixed bacterial liquid obtained in the step A is not less than 1 multiplied by 10 9 CFU/mL。
An application of a compound microbial agent for preventing and treating potato scab.
Comparative examples 1 to 9
Changing the volume ratio of each bacterial suspension in the mixed bacterial liquid, and setting a comparison example, namely: the mixed bacterial liquid consists of bacillus bailii bacterial suspension, bacillus aryabhattai bacterial suspension and bacillus circulans bacterial suspension, and the volume ratio of the three bacterial suspensions is as follows:
TABLE 1 volume ratio of bacterial suspensions in Mixed bacterial solutions
Experimental part
The inhibition effect of three strains on Streptomyces scab was verified:
preparing a streptomyces scab bacterial suspension: inoculating potato Streptomyces scab in ISP2 medium, culturing at 30deg.C for 7 days, scraping mature Streptomyces scab, and sterilizingIn water, the concentration is 1×10 8 The spore suspension of cfu/mL was ready for use.
100 mu L of Streptomyces scab spore suspension is uniformly coated on PDA solid culture medium, 6 mu L of bacterial suspension cultured by bacterial strain is sucked and dripped into the culture dish, the diameter of a bacteriostasis circle is measured after co-culture is carried out for 7d at 28-30 ℃, the ratio of the bacteriostasis diameter=the diameter of the bacteriostasis circle/the diameter of pathogenic bacteria multiplied by 100%, and the result is recorded.
TABLE 2 inhibition effect of three strains on Streptomyces scab
| Diameter/cm of inhibition zone | Antibacterial diameter ratio% | |
| Bacillus bailii | 5.12 | 60.52 |
| Bacillus aryabhattai | 4.05 | 56.45 |
| Bacillus circulans | 2.12 | 26.12 |
From the data in Table 1 and FIG. 1, it can be seen that the three strains of the invention have bacteriostasis to pathogenic bacteria and bacteriostatic effects: bacillus bailii > Bacillus aryabhattai > Bacillus circulans.
Potting experiment:
test bacterial agent: the microbial agents obtained in examples 1 to 3 (S1 to S3) and comparative examples 1 to 9 (S4 to 12) were diluted 600-fold and then applied.
Fourteen experiments were set up, three pots per group, and the results of each group averaged.
The test method comprises the following steps:
treatment group: filling vermiculite in flowerpots with the diameter of 20cm, transplanting 3 virus-free test-tube seedlings of potato varieties in each flowerpot, and culturing under the conditions that the illumination intensity is 50000lx and the photoperiod is L16h/D8 h. 10mL of spore suspension of Streptomyces scab is injected into the root of each seedling after 10d, and 30mL of the microbial inoculum (S1-S12) to be tested is evenly poured into each pot after 10d of culture.
Control group: as a blank control K1, a treatment without inoculating any bacteria was used, and only Streptomyces scab was injected as a positive control K2.
The disease index and the control effect are calculated through potato block grading, and the grading standard of scab is as follows:
disease grading standard: the potato peel is intact, has no disease spots and is grade 0; the area of the epidermoid plaque is more than 0 and less than 1/6, and is 1 grade; the area of the epidermoid plaque is more than 1/6 and less than 1/3, and is grade 2; the area of the epidermoid plaque is more than 1/3 and less than 1/2, and is grade 3; the area of the epidermoid plaque is more than 1/2 and is grade 4.
Incidence (%) = number of tubers diseased/number of tubers investigated x 100;
disease index = Σ (number of tubers of each disease level×representative value of the disease level)/(sum of investigation individuals×highest disease level) ×100%;
relative control (%) = (control disease index-treatment disease index)/control disease index x 100.
TABLE 3 potted plant experiment results
Field experiment:
experiments and 2022 are carried out in modern agriculture and technology experimental gardens in Linyi city and county of Shandong province in 5 months, and the tested varieties are as follows: potato N8, early crop conditions: potatoes, 40% infected with scab.
Treatment group:
the microbial agents obtained in examples 1 to 3 (S1 to S3) and comparative examples 1 to 9 (S4 to 12) were diluted 600-fold and then applied.
The test adopts a random block design, and the area of each cell is 24m 2 . The microbial inoculum is subjected to hole fertilization treatment on tubers at the time of sowing at 30 mL/plant, sowing in the last ten days of 5 months, field management is the same as local application, and harvesting in the last ten days of 8 months of the current year. Conventional fertilization: 2000kg/hm of base fertilizer application compound fertilizer (12-19-18) 2 。
Treatment group ck: conventional fertilization;
treatment group 1: conventional fertilization + the composite microbial agent prepared in example 1;
treatment group 2: conventional fertilization + the composite microbial agent prepared in example 2;
treatment group 3: conventional fertilization + the composite microbial agent prepared in example 3;
treatment group 4: conventional fertilization and the compound microbial agent prepared in comparative example 1;
treatment group 5: conventional fertilization and the compound microbial agent prepared in comparative example 2;
treatment group 6: conventional fertilization and the compound microbial agent prepared in comparative example 3;
treatment group 7: conventional fertilization and the compound microbial agent prepared in comparative example 4;
treatment group 8: conventional fertilization and the compound microbial agent prepared in comparative example 5;
treatment group 9: conventional fertilization and the compound microbial agent prepared in comparative example 6;
treatment group 10: conventional fertilization and the compound microbial agent prepared in comparative example 7;
treatment group 11: conventional fertilization and the compound microbial agent prepared in comparative example 8;
treatment group 12: conventional fertilization and the compound microbial agent prepared in comparative example 9;
agronomic shape determination:
plant height and stem thickness measurement: in the bud stage of the potato, 3 points are randomly taken from each treatment, 10 samples are continuously investigated at each point, and the plant height and the stem thickness are measured. The plant height is measured by a tape measure, and the linear distance from the root of the potato stem to the highest growing point of the potato is measured; the stem thickness is measured by a vernier caliper to measure the diameter of the thickest stem near the root of the potato.
Yield measurement during harvest: 3 points and 2m sample sections are randomly taken for each treatment to measure the yield of the potatoes.
Quality measurement: dry matter content is measured by dry weight method; the starch content was determined by iodine colorimetry.
TABLE 4 results of field experiments
| Yield t/hm 2 | Plant height cm | Thick stem mm | Dry matter% | Starch content% | |
| Example 1 | 60.99 | 62.36 | 10.69 | 20.12 | 19.56 |
| Example 2 | 61.69 | 62.11 | 10.78 | 20.98 | 19.45 |
| Example 3 | 61.11 | 62.89 | 10.87 | 21.57 | 20.78 |
| Comparative example 1 | 58.12 | 61.25 | 10.11 | 19.12 | 18.12 |
| Comparative example 2 | 57.47 | 61.02 | 10.04 | 19.01 | 18.05 |
| Comparative example 3 | 57.11 | 61.11 | 9.81 | 19.02 | 17.88 |
| Comparative example 4 | 55.89 | 60.56 | 9.56 | 18.12 | 16.25 |
| Comparative example 5 | 55.97 | 60.59 | 9.61 | 18.16 | 16.29 |
| Comparative example 6 | 56.04 | 60.69 | 9.62 | 18.39 | 16.55 |
| Comparative example 7 | 54.12 | 59.12 | 9.32 | 17.55 | 15.78 |
| Comparative example 8 | 54.98 | 59.69 | 9.39 | 17.59 | 15.88 |
| Comparative example 9 | 55.11 | 56.66 | 9.41 | 17.52 | 15.91 |
| ck | 52.11 | 58.47 | 9.25 | 15.78 | 11.55 |
From the data in tables 3-4, the microbial agent disclosed by the embodiment of the invention has good prevention effect on potato scab, and can promote good development of potato plants and improve the yield and quality of potatoes. The microbial agent composition is changed to be 1-9 in comparative examples, and the synergistic balance effect among the three functional microbial strains is weakened, and the interaction promotion effect is weakened, so that the disease resistance and the promotion effect on the potatoes are reduced.
It should be noted that the above-mentioned embodiments are merely some, but not all embodiments of the preferred mode of carrying out the invention. It is evident that all other embodiments obtained by a person skilled in the art without making any inventive effort, based on the above-described embodiments of the invention, shall fall within the scope of protection of the invention.
Claims (9)
1. A composite microbial agent is characterized by comprising mixed bacterial liquid and a bacterial seed accelerator.
2. The composite microbial agent according to claim 1, wherein the solid-to-liquid ratio of the mixed bacterial liquid and the bacterial seed accelerator is 100mL:5-10g.
3. The compound microbial agent of claim 1, wherein the mixed bacterial liquid comprises bacillus beijerinckii (Bacillus velezensis) bacterial suspension, bacillus aryabhattai (Bacillus aryabhattai) bacterial suspension and bacillus circulans (Bacillus circulans) bacterial suspension, and the volume ratio of the three bacterial suspensions is 1:1:1.
4. The composite microbial agent according to claim 2, wherein bacillus belicus is purchased from the China general microbiological culture collection center, the preservation number is CGMCC No.1.12669, bacillus aryabhattai is purchased from the China general microbiological culture collection center, the preservation number is CGMCC No.1.16121, and bacillus circulans is purchased from the China general microbiological culture collection center, and the preservation number is CGMCC No.1.8819.
5. The compound microbial agent according to claim 1, wherein the strain promoter comprises glucose, whey powder, ascorbic acid and ammonium molybdate in a mass ratio of (40-50): 10-20): 2-5): 1-3.
6. A method for preparing the composite microbial agent according to any one of claims 1 to 5, comprising the following steps:
A. activating Bacillus bailii, bacillus aryabhattai and Bacillus circulans in LB solid culture medium, respectively, inoculating single colony into test tube of LB liquid culture medium, and culturing at 28-30deg.C and 180-200r/min to OD 600 =0.8, resulting in seed solution; inoculating the seed solution into 200mLLB liquid culture medium according to the amount of 1%, culturing for 12h at 28-30 ℃ at 180-200r/min, washing the seed solution with sterile PBS buffer solution for 3 times, and then re-suspending the seed solution to obtain three bacterial suspensions, and mixing the three bacterial suspensions according to the volume ratio of 1:1:1 to obtain mixed bacterial solution;
B. glucose, whey powder, ascorbic acid and ammonium molybdate are prepared according to a certain proportion, and are uniformly mixed to form a strain promoter;
C. packaging the mixed bacterial liquid and the bacterial accelerant respectively, wherein when the mixed bacterial liquid and the bacterial accelerant are used, the solid-liquid ratio is 100mL:5-10g, diluted 500-800 times and applied.
7. The method for preparing the composite microbial agent according to claim 6, wherein the preparation method of the LB liquid medium in the step A is as follows: mixing 1L of water, 10g of tryptone, 5g of yeast extract and 10g of NaCl until solute is dissolved, and regulating the pH to 7.0 by using 3-5mol/L of NaOH to obtain an LB liquid culture medium; the LB solid culture medium is obtained by adding 15-20g agar based on LB liquid culture medium.
8. The method for producing a composite microbial agent according to claim 6, wherein the number of viable bacteria in the mixed bacterial liquid obtained in the step A is not less than 1X 10 9 CFU/mL。
9. Use of a composite microbial agent according to any one of claims 1 to 5 for the control of potato scab.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410038813.3A CN117821331B (en) | 2024-01-10 | 2024-01-10 | Composite microbial agent and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410038813.3A CN117821331B (en) | 2024-01-10 | 2024-01-10 | Composite microbial agent and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117821331A true CN117821331A (en) | 2024-04-05 |
| CN117821331B CN117821331B (en) | 2024-08-16 |
Family
ID=90505991
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410038813.3A Active CN117821331B (en) | 2024-01-10 | 2024-01-10 | Composite microbial agent and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117821331B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118931807A (en) * | 2024-10-15 | 2024-11-12 | 山东中新农业发展有限公司 | A microbial agent for preventing and controlling soil-borne diseases of ginger, and its preparation method and application |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003277210A (en) * | 2002-03-22 | 2003-10-02 | Mitsui Chemicals Inc | Plant disease controlling agent using genus bacillus bacterium and controlling method using the agent |
| CN106396864A (en) * | 2016-08-29 | 2017-02-15 | 佛山市艳晖生物科技有限公司 | Microbial fertilizer for control of potato scab and preparation method thereof |
| JP2019038788A (en) * | 2017-08-28 | 2019-03-14 | 片倉コープアグリ株式会社 | Microbial-containing control material and control method for potato scab |
| CN110291059A (en) * | 2017-01-12 | 2019-09-27 | 喜施倍全球股份有限公司 | Microbe soil reinforcing agent |
| CN111892440A (en) * | 2020-08-12 | 2020-11-06 | 宝鸡市星星协力生物有限公司 | Microbial fertilizer for preventing and treating potato scab and preparation method and use method thereof |
| CN116376784A (en) * | 2023-05-23 | 2023-07-04 | 临沂市农业科学院 | Microbial flora for preventing and treating peanut root rot |
| CN116536179A (en) * | 2022-12-12 | 2023-08-04 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | Separation and identification and fermentation process of potato scab antagonistic bacteria |
| CN116836896A (en) * | 2023-09-04 | 2023-10-03 | 山东植知源生态工程有限公司 | A kind of microbial inoculant for preventing and treating cotton fusarium wilt and preparation method thereof |
| CN116948909A (en) * | 2023-08-01 | 2023-10-27 | 甘肃省畜牧兽医研究所 | Livestock manure organic fertilizer starter and preparation method thereof |
-
2024
- 2024-01-10 CN CN202410038813.3A patent/CN117821331B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003277210A (en) * | 2002-03-22 | 2003-10-02 | Mitsui Chemicals Inc | Plant disease controlling agent using genus bacillus bacterium and controlling method using the agent |
| CN106396864A (en) * | 2016-08-29 | 2017-02-15 | 佛山市艳晖生物科技有限公司 | Microbial fertilizer for control of potato scab and preparation method thereof |
| CN110291059A (en) * | 2017-01-12 | 2019-09-27 | 喜施倍全球股份有限公司 | Microbe soil reinforcing agent |
| JP2019038788A (en) * | 2017-08-28 | 2019-03-14 | 片倉コープアグリ株式会社 | Microbial-containing control material and control method for potato scab |
| CN111892440A (en) * | 2020-08-12 | 2020-11-06 | 宝鸡市星星协力生物有限公司 | Microbial fertilizer for preventing and treating potato scab and preparation method and use method thereof |
| CN116536179A (en) * | 2022-12-12 | 2023-08-04 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | Separation and identification and fermentation process of potato scab antagonistic bacteria |
| CN116376784A (en) * | 2023-05-23 | 2023-07-04 | 临沂市农业科学院 | Microbial flora for preventing and treating peanut root rot |
| CN116948909A (en) * | 2023-08-01 | 2023-10-27 | 甘肃省畜牧兽医研究所 | Livestock manure organic fertilizer starter and preparation method thereof |
| CN116836896A (en) * | 2023-09-04 | 2023-10-03 | 山东植知源生态工程有限公司 | A kind of microbial inoculant for preventing and treating cotton fusarium wilt and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 李彩虹;杨志辉;张岱;赵冬梅;潘阳;朱杰华;: "马铃薯枯萎病拮抗菌的筛选与鉴定", 江苏农业科学, no. 13, 16 July 2018 (2018-07-16), pages 92 - 95 * |
| 石莹莹;赵盼;宋双伟;熊悯梓;莫乘宝;仲乃琴;: "马铃薯疮痂病拮抗菌YN-2-2的分离与鉴定", 微生物学通报, no. 08, 4 June 2020 (2020-06-04), pages 2425 - 2435 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118931807A (en) * | 2024-10-15 | 2024-11-12 | 山东中新农业发展有限公司 | A microbial agent for preventing and controlling soil-borne diseases of ginger, and its preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117821331B (en) | 2024-08-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111944716B (en) | Special compound microbial agent for tobacco seedling culture and preparation method and application thereof | |
| CN115340968B (en) | New application of pseudomonas microphylla and method thereof, pseudomonas microphylla 21.1.9.2-14 and products thereof | |
| CN108277177B (en) | Streptomyces microflavus solid fermentation medium, preparation method and fermentation method thereof, fermentation product, biocontrol product and application | |
| CN112342173A (en) | Bacillus belgii and application thereof | |
| CN117004508B (en) | Bacillus composite microbial inoculum and application thereof in citrus disease prevention and control | |
| CN112322502B (en) | Microbial inoculum prepared from Trichoderma africanum Ta97 and application thereof in preventing and treating continuous cropping diseases | |
| CN117821331B (en) | Composite microbial agent and preparation method and application thereof | |
| CN110938559A (en) | Compound microbial agent for preventing and treating soil-borne diseases, preparation method and application thereof in summer corn cultivation | |
| CN115975821B (en) | A kind of African Trichoderma harzianum Tr35 and its application | |
| CN113278563A (en) | Special soil improvement microbial agent for strawberries | |
| CN109315265B (en) | A kind of preparation method and application of Chaetomium globosa biological seedling substrate | |
| CN118685322B (en) | Streptomyces oryzae, bacterial agents and their applications | |
| CN109456900B (en) | Composite biological preparation and application thereof | |
| CN118165892B (en) | Pseudomonas composite microbial agent and application thereof | |
| CN112481161A (en) | Microbial agent for promoting wheat growth and wheat breeding method thereof | |
| CN112715301A (en) | Method for promoting rice growth and repelling pests by utilizing Isaria javanicus | |
| CN116590155B (en) | Paenispira rosea strain, microbial agent, preparation method and application | |
| Sutthisa et al. | Development of Trichoderma formulation and application to control durian anthracnose disease | |
| CN114931150B (en) | Cultivation method for improving medicinal quality of cyclocarya paliurus leaves by using trichoderma asperellum | |
| CN113549557B (en) | Trichoderma composite biological agent for preventing and treating blight of casserole watermelon and application thereof | |
| CN111471620B (en) | Biocontrol bacterium BV03, application thereof, microbial inoculum and preparation method of microbial inoculum | |
| CN114634878A (en) | Compound microbial preparation and application thereof in preventing and treating root-knot nematodes | |
| CN112674116A (en) | Compound microbial agent for preventing and treating scab of crops and preparation and use methods thereof | |
| CN114149935A (en) | Lysobacter enzymogenes and application thereof | |
| CN115895972B (en) | A compound bacterial agent and its application in the prevention and control of tobacco soil-borne diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |