CN114686238A - Preparation method and application of multifunctional soil microorganism conditioning microbial inoculum - Google Patents
Preparation method and application of multifunctional soil microorganism conditioning microbial inoculum Download PDFInfo
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- CN114686238A CN114686238A CN202210172561.4A CN202210172561A CN114686238A CN 114686238 A CN114686238 A CN 114686238A CN 202210172561 A CN202210172561 A CN 202210172561A CN 114686238 A CN114686238 A CN 114686238A
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- 239000002689 soil Substances 0.000 title claims abstract description 166
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 56
- 230000003750 conditioning effect Effects 0.000 title claims abstract description 54
- 244000005700 microbiome Species 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 75
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- 238000000034 method Methods 0.000 claims abstract description 12
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- 239000003795 chemical substances by application Substances 0.000 claims description 4
- GCLGEJMYGQKIIW-UHFFFAOYSA-H sodium hexametaphosphate Chemical group [Na]OP1(=O)OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])O1 GCLGEJMYGQKIIW-UHFFFAOYSA-H 0.000 claims description 4
- 235000019982 sodium hexametaphosphate Nutrition 0.000 claims description 4
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- 239000000126 substance Substances 0.000 description 19
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 18
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 18
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- 239000000243 solution Substances 0.000 description 11
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 description 9
- DOIRQSBPFJWKBE-UHFFFAOYSA-N dibutyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCCCC DOIRQSBPFJWKBE-UHFFFAOYSA-N 0.000 description 8
- 230000009467 reduction Effects 0.000 description 7
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- SIXWIUJQBBANGK-UHFFFAOYSA-N 4-(4-fluorophenyl)-1h-pyrazol-5-amine Chemical compound N1N=CC(C=2C=CC(F)=CC=2)=C1N SIXWIUJQBBANGK-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
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- MNSPJHASFCDXMX-UHFFFAOYSA-N 1,4-ditert-butylcyclohexa-2,4-dien-1-ol Chemical compound CC(C)(C)C1=CCC(O)(C(C)(C)C)C=C1 MNSPJHASFCDXMX-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/40—Soil-conditioning materials or soil-stabilising materials containing mixtures of inorganic and organic compounds
-
- 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
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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
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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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- 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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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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Abstract
The invention provides a preparation method and application of a multifunctional soil microorganism conditioning microbial inoculum, and belongs to the technical field of tobacco planting and soil treatment. The preparation method comprises the following steps: s1, preparing a composite biological control agent; s2, compounding a multifunctional microbial soil conditioning microbial inoculum. The prepared multifunctional soil microorganism conditioning microbial inoculum is applied to the quality evaluation of tobacco plants and tobacco leaves, and the application method comprises the following steps: s1, setting an experimental group and a control group; s2, sampling soil; s3, sampling tobacco leaves; s4, detecting a soil microflora; s5, investigating and analyzing the disease condition of the soil-borne disease; and S6, evaluating the quality of the flue-cured tobacco plants and the tobacco leaves. The invention solves the problems that the existing soil conditioner has incomplete improvement effect and the effect can not be visually reflected by the quality of tobacco leaves, and has the advantages of effectively reducing toxic substances in soil and improving the quality of the tobacco leaves.
Description
Technical Field
The invention relates to the technical field of tobacco planting and soil treatment, in particular to a preparation method and application of a multifunctional soil microorganism conditioning microbial inoculum.
Background
Tobacco is an important leaf economic crop, and is a plant of the genus Nicotiana of the family Solanaceae, an annual or limited perennial herb, with glandular hair overall and strong roots. The stem is 0.7-2 m high, and the base is slightly lignified. Tobacco is native to south America. China is widely cultivated in the north and south provinces. The tobacco is a temperature-loving crop, the response to the temperature is sensitive, and the influence of different temperature conditions on the quality and the yield of the tobacco is large. The requirement of high-quality tobacco on temperature in the growth period is lower in the early stage and higher in the later stage.
The soil environment has non-negligible influence on the growth of tobacco and the quality of the final tobacco leaves, and the environment suitable for the growth of tobacco comprises the following conditions:
(1) the surface soil is loose light soil, the core soil is compact soil, the water and fertilizer retention and water drainage and ventilation functions of the soil are good, the early and middle growth and the later growth of tobacco are facilitated, the tobacco can be yellow, and the yield and the quality are good. The soil texture of the high-quality tobacco zone is sandy loam to medium loam, the soil volume-weight scale value is 1.1-1.4 g per cubic centimeter, the soil total porosity scale value is 47.3% -56.9%, and the field water holding capacity scale of the soil is 23.9% -28.9%;
(2) the pH value of the soil is proper, and the pH value of the soil which is most suitable for the growth of tobacco plants is 5.5-6.5;
(3) appropriate soil fertility: different types of tobacco have different requirements on soil fertility. It is considered that flue-cured tobacco requires moderate organic matter content in soil, aromatic tobacco requires poor soil and low nitrogen content, and burley tobacco requires fertile soil.
However, the existing soil has low organic matter content and poor physical structure character, and in addition, chemical fertilizers and pesticides are excessively applied for a long time, harmful substance residues in the soil are serious, nutrient supply is imbalance, and the soil ecology is unbalanced, so that the requirement of healthy and continuous production of tobacco on the soil cannot be met, and therefore, the soil conditioner for tobacco growth is transported and grows.
The soil conditioner mainly comprises a natural conditioner, a synthetic conditioner and a natural-synthetic copolymer conditioner, and a biological conditioner, but the four soil conditioners have the problem of incomplete improvement effect, and the effect cannot be intuitively reflected through the quality of tobacco leaves.
Disclosure of Invention
The invention solves the technical problems that: the existing soil conditioner has the problem of incomplete improvement effect, and the effect cannot be intuitively reflected through the quality of tobacco leaves.
In order to solve the problems, the technical scheme of the invention is as follows:
a preparation method of a multifunctional soil microorganism conditioning microbial inoculum comprises the following steps:
s1, preparing the composite biological control bacterial agent, which comprises the following steps:
s1-1, preparing four 100ml LB culture media, the LB culture media comprises the following components: 10.0g/L peptone, 5.0g/L yeast extract powder, 10.0g/L peptone, the pH value range of LB culture medium is 7.0-7.2,
s1-2, respectively inoculating 100ul of amylolytic bacillus liquid, bacillus subtilis liquid and amylolytic bacillus liquid into four 100ml LB culture media, culturing for 12 hours at 37 ℃ in a shaking table, respectively diluting into bacterial liquids with bacterial concentration of 2 hundred million viable bacteria/g, mixing the bacterial liquids into mixed bacterial liquids according to the volume ratio of 1:1:1:1, then carrying out centrifugal operation to remove supernatant, collecting thalli, drying in a 60 ℃ oven, and then sieving through a 100-mesh sieve to obtain the composite biocontrol microbial inoculum;
s2, compounding a multifunctional microbial soil conditioning microbial inoculum, which specifically comprises the following steps:
s2-1, preparing four 100ml LB culture media, the LB culture media comprises the following components: 10.0g/L peptone, 5.0g/L yeast extract powder, 10.0g/L peptone, the pH value range of LB culture medium is 7.0-7.2,
s2-2, respectively inoculating 100ul of composite biocontrol microbial inoculum, potassium bacteria solution, phosphate-solubilizing bacteria solution and growth-promoting bacteria solution into four 100ml LB culture media, culturing for 12h at 37 ℃ in a shaking table, respectively diluting into bacteria solution with the bacteria concentration of 2 hundred million viable bacteria/g, mixing into mixed bacteria solution according to the volume ratio of 1:1:1:1,
and S2-3, mixing the mixed bacterial liquid and the white carbon black in a mass ratio of 5:1, drying the mixed bacterial liquid and the white carbon black in an oven at the temperature of 60 ℃, sieving the dried mixed bacterial liquid with a 100-mesh sieve to obtain a dry powder microbial inoculum master powder, adding a dispersing agent, a wetting agent and a stabilizing agent according to a certain mass ratio after obtaining the dry powder microbial inoculum master powder, and preparing the compound multifunctional microbial soil conditioning microbial inoculum after uniformly mixing.
Further, the centrifugation speed of the centrifugation operation in the step S1-2 is 12000rpm/min, the centrifugation time is 2min, and the high-speed centrifugation can quickly achieve the effect of separating substances.
Further, in step S2-3, the dispersant is sodium hexametaphosphate accounting for 2.00 wt% of the mass of the multifunctional microbial soil conditioning microbial inoculum, the wetting agent is sodium butylnaphthalene sulfonate accounting for 2.00 wt% of the mass of the multifunctional microbial soil conditioning microbial inoculum, the stabilizer is glycerin accounting for 1.00 wt% of the mass of the multifunctional microbial soil conditioning microbial inoculum, the sodium hexametaphosphate dispersant is an auxiliary agent for improving and improving the dispersibility of solid or liquid materials, and is helpful for crushing particles and preventing the crushed particles from coagulating to keep the dispersion stable, the sodium butylnaphthalene sulfonate has good wetting, diffusing and emulsifying capabilities, and the glycerin is easily soluble in water and common vegetable oil, animal oil and mineral oil, and is an ideal material for an emulsion stabilizer.
Furthermore, the multifunctional soil microorganism conditioning microbial inoculum is applied to the quality evaluation of tobacco plants and tobacco leaves, and the advantages of the multifunctional soil microorganism conditioning microbial inoculum can be objectively embodied.
The application of the multifunctional soil microorganism conditioning microbial inoculum comprises the following steps:
s1, selecting two groups of tobacco planting soil to serve as an experimental group and a control group respectively, applying a multifunctional soil microorganism conditioning microbial inoculum to the experimental group, wherein the application amount is 1 kg/mu of dry powder microbial inoculum, the application method comprises the steps of watering and irrigating roots after transplanting, and the control group does not apply the multifunctional soil microorganism conditioning microbial inoculum;
s2, soil sampling: after the tobacco leaves are harvested, 0-20cm rhizosphere soil samples are respectively adopted by an experimental group and a control group according to a 5-point method, surface soil is shoveled off during sampling, 0.5kg of samples are taken, and the samples are refrigerated and stored to be tested;
s3, tobacco leaf sampling: respectively selecting 1kg of baked and modulated C3F-grade tobacco leaves from an experimental group and a control group;
s4, soil microflora detection, which comprises the following steps:
s4-1, strain culture: culturing soil bacteria with beef extract peptone agar culture medium, culturing fungi with martin culture medium, culturing actinomycetes with improved Gao's No. 1 culture medium,
s4-2, preparing a soil suspension: collecting a 0-20cm soil sample, weighing 1g soil sample for preparing a soil suspension, dividing the prepared soil suspension into three parts, and respectively diluting the three parts by 10 times in accordance with the suitable environments of soil bacteria, actinomycetes and mildews, wherein the dilution multiple of the soil suspension corresponding to the soil bacteria is 10-4-10-6The dilution multiple of soil suspension corresponding to actinomycetes is 10-3-10-5The dilution multiple of soil suspension corresponding to the mould is 10-2-10-4Repeating the dilution for 3 times, respectively inoculating the three soil suspensions on the culture medium corresponding to the strains,
s4-3, after mixed bacteria inoculation suspension, inverting the three culture mediums to a incubator for culture, culturing bacteria at the temperature of 36-37 ℃ for 1 day, culturing actinomycetes and fungi at the temperature of 24-26 ℃ for 4 days,
s4-4, after the three types of strains are cultured, respectively taking out the plates, counting bacterial colonies appearing on the plates, and respectively calculating the average values of bacterial colonies of soil bacteria, actinomycetes and fungi;
s5, investigation and analysis of the disease condition of soil-borne diseases: from the 35 th day after the tobacco plant is transplanted, soil-borne diseases are investigated once every 5 days until topping is finished, and the morbidity, disease index and relative prevention and control effect are calculated;
s6, growth and development of the flue-cured tobacco plants, yield and output value investigation and tobacco sensory quality evaluation.
In step S3, C3F is the tobacco leaf, C represents the middle leaf of the upper, middle and lower parts of the cured tobacco leaf, F represents the orange color of the cured tobacco leaf, and 3 represents the quality grade of the tobacco leaf. C3F is used as a common experimental part and can integrally reflect the quality of flue-cured tobacco.
Preferably, in step S5, the formula for calculating the incidence rate is:
the incidence rate is (number of diseased plants/total number of investigated plants) × 100%.
The calculation of the morbidity can evaluate the effect of the multifunctional soil microorganism conditioning microbial inoculum from the growth level of the plant.
Preferably, in step S5, the formula for calculating the disease index is:
the disease index [ ∑ (diseased plant or leaf number at each level × the disease grade value)/(total investigated plant number or leaf number × the highest grade value) ] × 100%.
The calculation of disease indexes can evaluate the effect of the multifunctional soil microorganism conditioning microbial inoculum from the disease level of diseased plants.
Preferably, in step S5, the formula for calculating the relative control effect is:
relative control effect is [ (control disease index-treatment disease index)/control disease index ] × 100%.
And the calculation of the relative prevention and treatment effect can evaluate the effect of the multifunctional soil microorganism conditioning microbial inoculum from the aspect of disease prevention and treatment.
Preferably, step S6 specifically includes: according to the sensory evaluation method of the quality, style and characteristics of the tobacco leaves, the internal quality of the tobacco leaf raw materials is evaluated through human smell and taste, and the evaluation on the effect of the multifunctional soil microorganism conditioning fungicide can be made from a human determination level.
Further preferably, the evaluation factors of step S6 include: the quality and quantity of the aroma and the comfort degree of the taste can evaluate the effect of the multifunctional soil microorganism conditioning microbial inoculum from the tobacco leaf quality level.
The invention has the beneficial effects that:
(1) the invention provides the processes of configuration, application and multi-aspect evaluation of a multifunctional soil microbial conditioning fungicide, the prepared multifunctional soil microbial conditioning fungicide can effectively reduce the contents of catechol, phthalic acid, p-hydroxybenzoic acid, butanol and dibutyl phthalate in rhizosphere soil, and particularly has a more obvious removing effect on two acid substances, namely phthalic acid and p-hydroxybenzoic acid, which have larger self-toxicity hazards, and is beneficial to the soil to meet the requirement of healthy and continuous production of tobacco;
(2) the multifunctional soil microorganism conditioning microbial inoculum prepared by the invention can reduce the quantity of soil molds by 43.75%, can obviously improve the metabolism and carbon source utilization activity of rhizosphere soil microorganisms, and improves the diversity (Shannon index) of the soil microorganisms, the relative density (Shannon homogeneity index) of various species in a community and the carbon source utilization intensity thereof, thereby showing that the structure of a soil microorganism area system is better improved;
(3) the self-made conditioner has a certain effect of preventing and treating bacterial wilt, the application of the soil conditioner can improve the root activity of tobacco plants and can obviously improve the yield and output value of tobacco leaves, wherein the output value of the tobacco leaves is increased by 26.97%, the hierarchical structure of the tobacco leaves is also better improved, and the sensory quality of the tobacco leaves is obviously improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. 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.
The terminology used in the embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the examples of the present invention and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise, and "a plurality" typically includes at least two.
It should be understood that although the terms first, second, third, etc. may be used to describe … … in embodiments of the present invention, these … … should not be limited to these terms. These terms are used only to distinguish … …. For example, the first … … can also be referred to as the second … … and similarly the second … … can also be referred to as the first … … without departing from the scope of embodiments of the present invention.
Example 1
A preparation method of a multifunctional soil microorganism conditioning microbial inoculum comprises the following steps:
s1, preparing the composite biological control bacterial agent, which comprises the following steps:
s1-1, preparing four 100ml LB culture media, the components and the content of the LB culture media are: 10.0g/L peptone, 5.0g/L yeast extract powder, 10.0g/L peptone, the pH value range of LB culture medium is 7.0-7.2,
s1-2, respectively inoculating 100ul of amylolytic bacillus liquid, bacillus subtilis liquid and amylolytic bacillus liquid into four 100ml LB culture media, carrying out shake cultivation for 12h at 37 ℃, respectively diluting the two bacteria liquids into bacteria liquid with the bacteria concentration of 2 hundred million viable bacteria/g, mixing the bacteria liquids into mixed bacteria liquid according to the volume ratio of 1:1:1:1, carrying out centrifugal operation to remove supernatant, collecting bacteria, drying in a 60 ℃ oven, and then sieving with a 100-mesh sieve to obtain the composite biocontrol microbial inoculum;
s2, compounding a multifunctional microbial soil conditioning microbial inoculum, which specifically comprises the following steps:
s2-1, preparing four 100ml LB culture media, the components and the content of the LB culture media are: 10.0g/L peptone, 5.0g/L yeast extract powder, 10.0g/L peptone, the pH value range of LB culture medium is 7.0-7.2,
s2-2, respectively inoculating 100ul of composite biocontrol microbial inoculum, potassium bacteria solution, phosphate-solubilizing bacteria solution and growth-promoting bacteria solution into four 100ml LB culture media, culturing for 12h at 37 ℃ in a shaking table, respectively diluting into bacteria solution with the bacteria concentration of 2 hundred million viable bacteria/g, mixing into mixed bacteria solution according to the volume ratio of 1:1:1:1,
and S2-3, mixing the mixed bacterial liquid and the white carbon black in a mass ratio of 5:1, drying the mixed bacterial liquid and the white carbon black in an oven at the temperature of 60 ℃, sieving the dried mixed bacterial liquid with a 100-mesh sieve to obtain a dry powder microbial inoculum master powder, adding a dispersing agent, a wetting agent and a stabilizing agent according to a certain mass ratio after obtaining the dry powder microbial inoculum master powder, and preparing the compound multifunctional microbial soil conditioning microbial inoculum after uniformly mixing.
The centrifugation speed of the centrifugation operation in the step S1-2 is 12000rpm/min, and the centrifugation time is 2 min.
In the step S2-3, the dispersant is sodium hexametaphosphate accounting for 2.00 wt% of the mass of the multifunctional microbial soil conditioning microbial inoculum, the wetting agent is sodium butylnaphthalenesulfonate accounting for 2.00 wt% of the mass of the multifunctional microbial soil conditioning microbial inoculum, and the stabilizer is glycerin accounting for 1.00 wt% of the mass of the multifunctional microbial soil conditioning microbial inoculum.
Example 2
The application of the multifunctional soil microorganism conditioning fungicide is applied to the quality evaluation of tobacco plants and tobacco leaves, and comprises the following steps:
s1, selecting two groups of tobacco planting soil to be respectively used as an experimental group and a control group, wherein the experimental group applies the multifunctional soil microorganism conditioning fungicide with the application amount of 1 kg/mu of dry powder fungicide, the application method is that the multifunctional soil microorganism conditioning fungicide is added with water to irrigate roots after transplanting, and the control group does not apply the multifunctional soil microorganism conditioning fungicide;
s2, soil sampling: after the tobacco leaves are harvested, 0-20cm rhizosphere soil samples are respectively adopted by an experimental group and a control group according to a 5-point method, surface soil is shoveled off during sampling, 0.5kg of samples are taken, and the samples are refrigerated and stored to be tested;
s3, tobacco leaf sampling: respectively selecting 1kg of baked and modulated C3F-grade tobacco leaves from an experimental group and a control group;
s4, soil microflora detection, which comprises the following steps:
s4-1, strain culture: culturing soil bacteria with beef extract peptone agar culture medium, culturing fungi with martin culture medium, culturing actinomycetes with improved Gao's No. 1 culture medium,
s4-2, preparing a soil suspension: collecting a 0-20cm soil sample, weighing 1g soil sample for preparing a soil suspension, dividing the prepared soil suspension into three parts, and respectively diluting the three parts by 10 times in accordance with the suitable environments of soil bacteria, actinomycetes and mildews, wherein the dilution multiple of the soil suspension corresponding to the soil bacteria is 10-4-10-6The dilution multiple of soil suspension corresponding to actinomycetes is 10-3-10-5The dilution multiple of soil suspension corresponding to the mould is 10-2-10-4Repeating the dilution for 3 times, respectively inoculating the three soil suspensions on the culture medium corresponding to the strains,
s4-3, after mixed bacteria inoculation suspension, inverting the three culture mediums to a incubator for culture, culturing bacteria at the temperature of 36-37 ℃ for 1 day, culturing actinomycetes and fungi at the temperature of 24-26 ℃ for 4 days,
s4-4, after the three types of strains are cultured, respectively taking out the plates, counting bacterial colonies appearing on the plates, and respectively calculating the average values of bacterial colonies of soil bacteria, actinomycetes and fungi;
s5, investigation and analysis of the disease condition of soil-borne diseases: investigating soil-borne diseases once every 5 days from the 35 th day after the tobacco plants are transplanted until topping is finished, calculating the morbidity, disease index and relative prevention and treatment effect,
wherein, the formula for calculating the incidence rate is as follows:
the incidence rate is (number of diseased plants/total number of investigated plants) × 100%,
the formula for calculating the disease index is:
disease index [ ∑ (number of diseased plants or leaves at each stage x the disease value)/(total number of investigated plants or leaves x highest value) ] × 100%,
the formula for calculating the relative control effect is as follows:
relative prevention and treatment effect is [ (control disease index-treatment disease index)/control disease index ] × 100%;
s6, growth and development of flue-cured tobacco plants, yield value investigation and sensory quality evaluation of tobacco leaves, which specifically comprise the following steps: the tobacco leaf raw materials are subjected to internal quality evaluation through human smell and taste according to a tobacco leaf quality, style and characteristic sensory evaluation method, and evaluation factors comprise: quality and quantity of fragrance, and comfort degree of taste.
Examples of the experiments
The experimental example is based on the analysis of the influence of the multifunctional soil microorganism conditioning microbial inoculum of the example 1 and the example 2 on soil allelochemicals after the application of the microbial inoculum to the soil.
1. Influence on rhizosphere soil chemical susceptibility autotoxic substances
Fertilizing the experimental group soil according to the application amount of the multifunctional soil microorganism conditioning fungicide of 1 kg/mu, not treating the control group soil, and then obtaining the soil chemical self-toxic substance content in the rhizosphere soil sample in the maturation period, wherein the results are shown in the following table:
TABLE 1 influence table of multifunctional soil microorganism conditioning fungicide on rhizosphere soil chemical-induced autotoxic substances
From the above contents, the multifunctional soil microorganism conditioning microbial inoculum can effectively reduce the contents of catechol, phthalic acid, p-hydroxybenzoic acid, butanol and dibutyl phthalate in rhizosphere soil. Wherein, for two acid substances with larger self-toxicity harm, phthalic acid and p-hydroxybenzoic acid can be respectively reduced by 18.97 percent and 45.71 percent after being applied.
2. Influence of different crop rotation modes on soil allelochemicals
Fertilizing soil according to the application amount of the multifunctional soil microbial conditioning agent of 1 kg/mu, planting different plants in the soil according to different crop rotation modes, and finally detecting the allelochemicals in the red soil in the soil, wherein the final results are shown in tables 2 and 3.
TABLE 22016 years soil allelochemicals table under different crop rotation modes
TABLE 32017 soil allelochemical substance table under different rotation modes
As is clear from tables 2 and 3, 13 types of chemosensory substances including 5 types of acids, lipids, aldehydes, phenols, and alcohols were detected in the tobacco-planting soil. The content of butanol is maximum and reaches over 100ug/kg, and the content of other substances is basically below 100 ug/kg.
The content of catechol is increased along with the extension of the continuous cropping age, and compared with 2016 in 2017, the content of the catechol is increased by 8.88 percent, and the content of phthalic acid, p-hydroxybenzoic acid, benzoic acid and 3-methoxy-4-hydroxybenzoic acid is increased by 9.81 percent, 69.86 percent, 5.55 percent and 157.07 percent respectively. By adopting different crop rotation, the pyrocatechol, the phthalic acid, the p-hydroxybenzoic acid, the benzoic acid and the 3-methoxyl-4-hydroxybenzoic acid in the soil are in a descending trend, the descending range is 4.65-59.09%, 26.88-66.58%, 2.87-46.50%, 4.98-81.22% and 36.00-66.57%.
3. Influence of winter ploughing depth on soil allelopathy substances
The soil was fertilized at a rate of 1 kg/mu of the multifunctional soil microbial conditioning inoculum, and the effect of the depth of winter cultivation on soil allelochemicals was studied in a manner of winter cultivation (rotary tillage and plowing), with the final results shown in table 4.
TABLE 4 soil allelochemical content scale under different depth conditions of winter cultivation
Under different winter ploughing depths, 4 kinds of acid substances such as phthalic acid, p-hydroxybenzoic acid, benzoic acid and terephthalic acid have consistent law under different winter ploughing depths, and the law is represented as follows: no winter ploughing is more than rotary tillage 20cm, ploughing is more than 20cm and ploughing is more than 40 cm. Among the 4 species, the terephthalic acid content was relatively high, and the phthalic acid content was low. The esters and aldehydes have different performances, wherein the diisobutyl phthalate has the lowest non-winter ploughing content and the highest ploughing content of 40 cm. The content of the p-hydroxybenzaldehyde is expressed as that the content is not ploughed in winter, more than 20cm by rotary tillage, more than 20cm by ploughing and more than 40cm by ploughing. The content of catechol is shown as follows in sequence: the maximum value of the content is 17.90 mu g/kg, the minimum value is 9.22 mu g/kg, and the variation coefficient is 36.35 percent.
4. Effect of application of Biomass charcoal on soil allelochemicals
Setting experimental group soil and control group soil, applying biomass with different dosages to the experimental group soil, not processing the control group soil, and then acquiring the soil chemical autotoxicity content in the rhizosphere soil sample in the maturation period, wherein the results are shown in the following table:
TABLE 5 soil allelochemical content table under biomass charcoal application
As can be seen from the table above, the application of the biomass charcoal can effectively reduce the amount of allelochemicals in the soil, and the contents of catechol, o-butyl phenol and p-di-tert-butyl phenol are reduced along with the increase of the amount of the biomass charcoal. Compared with a contrast, after the biomass charcoal is applied, the catechol is reduced by 17.86-62.60%, and the ortho-and para-di-tert-butylphenol is reduced by 20.70-62.60%; the reduction range of the phthalic acid is 18.46 to 47.40 percent, the reduction range of the p-hydroxybenzoic acid is 7.81 to 36.56 percent, the reduction range of the benzoic acid is 6.19 to 39.32 percent, the reduction range of the 3-methoxy-4-hydroxybenzoic acid is 33.00 to 39.19 percent, and the reduction range of the terephthalic acid is 1.02 to 21.48 percent; the content of dibutyl phthalate and diisooctyl phthalate, wherein the reduction range of dibutyl phthalate is 7.39-35.76%, and the reduction range of diisooctyl phthalate is 0.47-63.32%.
5. Influence of applying organic fertilizer on soil allelochemicals
Setting experimental group soil and control group soil, applying organic fertilizers (tobacco straw fertilizer and farmyard manure) to the experimental group soil, not processing the control group soil, and then obtaining the content of soil chemical self-toxic substances in a rhizosphere soil sample in a maturation period, wherein the results are shown in the following table:
TABLE 6 soil allelochemical substance content table under organic fertilizer application
From the table above, applying the organic fertilizer can effectively reduce the content of allelochemicals in the soil, wherein the content of allelochemicals such as phthalic acid, benzoic acid, p-hydroxybenzaldehyde and diisobutyl phthalate is shown as follows: compared with the control group, the contents of phthalic acid, benzoic acid, p-hydroxybenzaldehyde, diisobutyl phthalate and the like are respectively reduced by 79.74%, 50.33%, 47.53% and 88.20%. The content sequence of the sensates such as catechol, p-hydroxybenzoic acid and 3-methoxy-4-hydroxybenzoic acid is shown as follows: compared with the control group, the content of each substance is reduced by 70.81%, 26.61% and 65.70% respectively.
Claims (9)
1. A preparation method of a multifunctional soil microorganism conditioning microbial inoculum is characterized by comprising the following steps:
s1, preparing the composite biological control bacterial agent, which comprises the following steps:
s1-1, preparing four 100ml LB culture media, the LB culture media comprises the following components: 10.0g/L peptone, 5.0g/L yeast extract powder, 10.0g/L peptone, the pH value range of LB culture medium is 7.0-7.2,
s1-2, respectively inoculating 100ul of amylolytic bacillus liquid, bacillus subtilis liquid and amylolytic bacillus liquid into four 100ml LB culture media, carrying out shake cultivation for 12h at 37 ℃, respectively diluting the two bacteria liquids into bacteria liquid with the bacteria concentration of 2 hundred million viable bacteria/g, mixing the bacteria liquids into mixed bacteria liquid according to the volume ratio of 1:1:1:1, carrying out centrifugal operation to remove supernatant, collecting bacteria, drying in a 60 ℃ oven, and then sieving with a 100-mesh sieve to obtain the composite biocontrol microbial inoculum;
s2, compounding a multifunctional microbial soil conditioning microbial inoculum, which specifically comprises the following steps:
s2-1, preparing four 100ml LB culture media, the LB culture media comprises the following components: 10.0g/L peptone, 5.0g/L yeast extract powder, 10.0g/L peptone, the pH value range of LB culture medium is 7.0-7.2,
s2-2, respectively inoculating 100ul of composite biocontrol microbial inoculum, potassium-solubilizing bacterial liquid, phosphorus-solubilizing bacterial liquid and growth-promoting bacterial liquid into four 100ml LB culture media, culturing for 12h at 37 ℃ in a shaking table, respectively diluting into bacterial liquids with the bacterial concentration of 2 hundred million viable bacteria/g, mixing into mixed bacterial liquids according to the volume ratio of 1:1:1:1,
and S2-3, mixing the mixed bacterial liquid and the white carbon black, wherein the mass ratio of the mixed bacterial liquid to the white carbon black is 5:1, drying the mixed bacterial liquid and the white carbon black in an oven at the temperature of 60 ℃, sieving the dried bacterial liquid by a 100-mesh sieve to obtain a dry powder microbial inoculum mother powder, adding a dispersing agent, a wetting agent and a stabilizing agent according to a certain mass ratio after obtaining the dry powder microbial inoculum mother powder, and preparing the compound multifunctional microbial soil conditioning microbial inoculum after uniformly mixing.
2. The method for preparing a multifunctional soil microorganism conditioning microbial inoculum as claimed in claim 1, wherein the centrifugation speed of the centrifugation operation in the step S1-2 is 12000rpm/min, and the centrifugation time is 2 min.
3. The method according to claim 1, wherein in step S2-3, the dispersant is sodium hexametaphosphate accounting for 2.00 wt% of the multifunctional microbial soil conditioner, the wetting agent is sodium butylnaphthalene sulfonate accounting for 2.00 wt% of the multifunctional microbial soil conditioner, and the stabilizer is glycerol accounting for 1.00 wt% of the multifunctional microbial soil conditioner.
4. The application of the multifunctional soil microorganism conditioning fungicide prepared according to the method of claims 1-3 is characterized in that the multifunctional soil microorganism conditioning fungicide is applied to the quality evaluation of tobacco plants and tobacco leaves.
5. The application of the multifunctional soil microorganism conditioning microbial inoculum of claim 4, wherein the application method comprises the following steps:
s1, selecting two groups of tobacco planting soil to serve as an experimental group and a control group respectively, applying the multifunctional soil microorganism conditioning microbial inoculum to the experimental group, wherein the application amount is 1 kg/mu of dry powder microbial inoculum, the application method is that the roots are irrigated by adding water after transplanting, and the multifunctional soil microorganism conditioning microbial inoculum is not applied to the control group;
s2, soil sampling: after the tobacco leaves are harvested, 0-20cm rhizosphere soil samples are respectively adopted by an experimental group and a control group according to a 5-point method, surface soil is shoveled off during sampling, 0.5kg of samples are taken, and the samples are refrigerated and stored to be tested;
s3, tobacco leaf sampling: respectively selecting 1kg of baked and modulated C3F-grade tobacco leaves from an experimental group and a control group;
s4, soil microflora detection, which comprises the following steps:
s4-1, strain culture: culturing soil bacteria with beef extract peptone agar culture medium, culturing fungi with martin culture medium, culturing actinomycetes with improved Gao's No. 1 culture medium,
s4-2, preparing a soil suspension: collecting a 0-20cm soil sample, weighing 1g soil sample for preparing soil suspension, dividing the prepared soil suspension into three parts, and respectively diluting the three parts by 10 times in accordance with the suitable environments of soil bacteria, actinomycetes and mould, wherein the dilution multiple of the soil suspension corresponding to the soil bacteria is 10-4-10-6The dilution multiple of soil suspension corresponding to actinomycetes is 10-3-10-5The dilution ratio of soil suspension corresponding to the mould is 10-2-10-4Repeating the dilution for 3 times, respectively inoculating the three soil suspensions on the culture medium corresponding to the strains,
s4-3, after mixed bacteria inoculation suspension, inverting the three culture mediums to be cultured in an incubator, culturing bacteria at the temperature of 36-37 ℃ for 1 day, culturing actinomycetes and fungi at the temperature of 24-26 ℃ for 4 days,
s4-4, after the three types of strains are cultured, respectively taking out the plates, counting bacterial colonies appearing on the plates, and respectively calculating the average values of bacterial colonies of soil bacteria, actinomycetes and fungi;
s5, investigation and analysis of the disease condition of soil-borne diseases: from the 35 th day after the tobacco plant is transplanted, soil-borne diseases are investigated once every 5 days until topping is finished, and the morbidity, disease index and relative prevention and control effect are calculated;
s6, growth and development of the flue-cured tobacco plants, yield and output value investigation and tobacco sensory quality evaluation.
6. The application of the multifunctional soil microorganism conditioning microbial inoculum of claim 5, wherein in the step S5, the formula for calculating the incidence rate is as follows:
the incidence of disease (number of diseased plants/total number of investigated plants) × 100%.
7. The use of the multifunctional soil microorganism conditioning microbial inoculum of claim 5, wherein in the step S5, the formula for calculating the disease index is as follows:
the disease index [ Σ (number of diseased plants or leaves at each stage × the disease level value)/(total number of investigated plants or leaves × the highest level value) ] × 100%.
8. The application of the multifunctional soil microorganism conditioning microbial inoculum of claim 5, wherein in the step S5, the formula for calculating the relative control effect is as follows:
relative control effect is [ (control disease index-treatment disease index)/control disease index ] × 100%.
9. The application of the multifunctional soil microorganism conditioning microbial inoculum of claim 5, wherein the step S6 specifically comprises the following steps: and (3) performing internal quality evaluation on the tobacco raw materials through human smell and taste by referring to a tobacco quality, style and characteristic sensory evaluation method.
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