CN108707560B - Microbial liquid bacterial fertilizer and preparation method and application thereof - Google Patents

Microbial liquid bacterial fertilizer and preparation method and application thereof Download PDF

Info

Publication number
CN108707560B
CN108707560B CN201810498954.8A CN201810498954A CN108707560B CN 108707560 B CN108707560 B CN 108707560B CN 201810498954 A CN201810498954 A CN 201810498954A CN 108707560 B CN108707560 B CN 108707560B
Authority
CN
China
Prior art keywords
bacillus amyloliquefaciens
tea
liquid bacterial
microbial
fermentation
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.)
Active
Application number
CN201810498954.8A
Other languages
Chinese (zh)
Other versions
CN108707560A (en
Inventor
林斌
黄菊青
徐庆贤
郑怡
钱蕾
官雪芳
张臣心
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fujian Academy Of Agricultural Sciences Agricultural Product Processing Research Institute
Original Assignee
Institute of Agricultural Engineering Technology of Fujian Academy of Agricultural Sciences
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Institute of Agricultural Engineering Technology of Fujian Academy of Agricultural Sciences filed Critical Institute of Agricultural Engineering Technology of Fujian Academy of Agricultural Sciences
Priority to CN201810498954.8A priority Critical patent/CN108707560B/en
Publication of CN108707560A publication Critical patent/CN108707560A/en
Application granted granted Critical
Publication of CN108707560B publication Critical patent/CN108707560B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

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
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
    • C05F5/002—Solid waste from mechanical processing of material, e.g. seed coats, olive pits, almond shells, fruit residue, rice hulls
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C05G3/80—Soil conditioners
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G5/00—Fertilisers characterised by their form
    • C05G5/20—Liquid fertilisers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Mechanical Engineering (AREA)
  • Botany (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Environmental & Geological Engineering (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Soil Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Fertilizers (AREA)

Abstract

The invention discloses a microbial liquid bacterial fertilizer, which contains 1.8 multiplied by 10 viable bacteria8cfu/ml~2.2×108cfu/mL of fermentation broth of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) (II)Bacillus amyloliquefaciens) W208, the strain is preserved in the China general microbiological culture Collection center on 2016, 9 and 13 days, and the preservation number is as follows: CGMCC No. 13006. The invention also discloses a preparation method of the liquid bacterial fertilizer and application of the liquid bacterial fertilizer in tea production. The liquid bacterial fertilizer provided by the invention can obviously improve the soil environment, has good fertility, can obviously improve the tea yield, obviously reduce caffeine in tea and improve the tea taste, has the advantages of simple preparation method, low cost, good storage stability, simple use and environmental friendliness, and has important significance for promoting tea production and sustainable development of ecological tea gardens.

Description

Microbial liquid bacterial fertilizer and preparation method and application thereof
Technical Field
The invention relates to the technical field of agricultural fertilizers, in particular to a microbial liquid bacterial fertilizer and a preparation method and application thereof.
Background
As a main tea-producing country in the world, the tea consumption in China is rapidly increased in recent years, and reaches 161.32 ten thousand tons in 2013, which is 225 percent higher than 2001. Meanwhile, the global tea consumption increased from 298.58 ten thousand tons in 2001 to 482.1 ten thousand tons in 2013. The market demands a great deal of tea, and the large-area planting and the quality improvement of the tea are directly promoted. At present, China has become the fastest growing and most potential tea market worldwide. Improving the tea quality and increasing the tea yield are two key technical problems of improving the market competitiveness of tea products and increasing the economic benefits of tea manufacturing enterprises. Fertilizing is one of the most important agricultural measures for improving the yield and the quality of tea and promoting the sustainable development of tea gardens. In the prior art, a plurality of reports are reported on the research of tea fertilization, but the applied fertilizers mainly comprise chemical fertilizers, organic fertilizers and leguminous green fertilizers. However, with the blind use of chemical fertilizers in tea gardens and the increasingly prominent ecological problems and environmental problems caused by unreasonable fertilization modes, the development of environment-friendly and efficient fertilizers capable of promoting the sustainable development of tea gardens is of great importance and urgency.
The microbial fertilizer is a living fertilizer, is a novel fertilizer in twenty-first century, and is an organic microbial agent with low carbon, pure nature, no toxicity, no harm and no pollution. The biological fertilizer is mainly completed by the metabolism of the life activities of a large number of beneficial microorganisms, and has the functions of improving the soil fertility, increasing the number and the activity of the beneficial microorganisms in the soil, improving the soil properties, preventing the soil from hardening, improving the soil fertility, water retention and cold resistance, enhancing the disease resistance of crops, increasing organic matters in the soil, preventing the invasion of pathogenic bacteria, reducing the growth of plant diseases and insect pests, promoting the growth of crops, improving the yield of the crops, improving and reducing the quality of agricultural products and the like. Microbial fertilizers are classified into solid and liquid forms. The liquid microbial fertilizer is favored by the tea planting industry by the advantages of instant dissolution, uniformity, instant use, sprinkling irrigation and irrigation, difficult moisture absorption and caking and the like. However, the research on promoting tea planting by microbial fertilizers in the prior art generally focuses on promoting soil fertility and improving yield, and the research on promoting tea quality is relatively simple in function and relatively less. In addition, the technicians in the industry find that in the process of applying the liquid microbial fertilizer, the liquid fertilizer has the problems of poor stability, obvious reduction of the number of viable bacteria in the liquid bacterial fertilizer along with the increase of storage time and the reduction of the efficacy of the liquid bacterial fertilizer.
Disclosure of Invention
The invention aims to provide a microbial liquid bacterial fertilizer and a preparation method and application thereof, and aims to solve the problems of single function and poor storage stability of the conventional microbial liquid bacterial fertilizer.
The purpose of the invention is realized by the following technical scheme: a liquid bacterial fertilizer for microbes is prepared from the bacterial fertilizer containing viable bacteria (1.8X 10)8cfu/ml～2.2×108cfu/mL fermentation liquor of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) W208, wherein the strain is preserved in the general microbiological center of China Committee for culture Collection of microorganisms in 2016, 9 and 13 days, and the preservation numbers are as follows: CGMCC No. 13006. The fermentation broth is prepared by inoculating bacillus amyloliquefaciens in a fermentation culture medium and fermenting at 30-40 ℃; the fermentation medium is a liquid culture medium which is prepared by taking starch wastewater of a potato starch factory as a raw material, adding 10-30 g/L of soybean meal, adjusting the pH value to 6.0-8.0 and sterilizing.
Organic acid can be added into the liquid bacterial manure to increase the storage stability of the liquid bacterial manure; the adding mass of the organic acid is 0.01-2.0% of the mass of the liquid bacterial manure; preferably, the organic acid is any one of formic acid, acetic acid or citric acid. The viable count of the liquid bacterial manure added with the organic acid can be stably maintained for 3 months at normal temperature.
The strain morphology characteristics of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) W208 are as follows: the cells are rod-shaped, have the length of 1.0-2.0 mu m and the width of 0.3-0.6 mu m, have flagella and are gram-positive; after culturing for 48 hours on an LB plate culture medium, the colony is raised, unsmooth, opaque, milky white and dendritic at the edge; the growth of the material is suitable for the pH value of 6.0-8.0 and the suitable temperature of 30-40 ℃.
A preparation method of a microbial liquid bacterial fertilizer comprises the following steps:
(a) inoculating the bacillus amyloliquefaciens strain into an LB culture medium, and carrying out shaking culture for 24 hours at 180r/min in a constant-temperature shaking table at 30-40 ℃ to prepare liquid seeds; the bacillus amyloliquefaciens is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) W208, and the strain is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms in 2016, 9 and 13 days, wherein the preservation numbers are as follows: CGMCC No. 13006;
(b) inoculating the liquid seeds into a fermentation medium in a fermentation tank according to the inoculation amount of 5% by mass and volume, aerating, fermenting for 70-74 hours at 30-40 ℃, supplementing the fermentation medium in the fermentation process, wherein the viable count of the bacillus amyloliquefaciens in the liquid to be fermented is 1.8 multiplied by 108cfu/ml～2.2×108And (5) collecting fermentation liquor when cfu/mL, wherein the fermentation liquor is the microbial liquid bacterial manure.
In order to increase the efficacy stability of the microbial liquid bacterial manure, organic acid is added into the fermentation liquor; the adding mass of the organic acid is 0.01-2.0% of the mass of the fermentation liquor, and the preferable mass is 0.05%; the organic acid is one of formic acid, acetic acid or citric acid; the viable count of the liquid bacterial manure added with the organic acid can be stably kept for 3 months at normal temperature (15-25 ℃).
The LB culture medium comprises: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, pH value of 6.0-8.0, and sterilizing for 20min at 121.
The fermentation medium is a liquid culture medium prepared by taking starch wastewater of a potato starch factory as a raw material, adding 10-30 g/L of soybean meal, adjusting the pH value to 6.0-8.0, and sterilizing at 121 ℃ for 20 min. The COD of the starch wastewater is 18000-22000 mg/L.
The fermentation culture medium is supplemented for the first time in 24h of fermentation, the supplementation volume is 40-60% of the volume of the original fermentation liquid, and the fermentation culture medium is supplemented for the second time in 56h of fermentation, and the supplementation volume is 20-40% of the volume of the fermentation liquid before supplementation.
Experiments prove that the microbial liquid bacterial fertilizer provided by the invention can improve the tea yield, the tea quality, the soil environment and the microbial flora, and can be applied to tea production.
In tea planting, the application method of the microbial liquid bacterial fertilizer comprises the following steps: before use, the pH value of the fermented product is adjusted to 7.0 by using a sodium hydroxide solution with the concentration of 6mol/L, and the fermented product is diluted by 50 times by using the fermentation medium, and the recommended application rate is 1500L/mu.
The invention prepares the microbial liquid bacterial fertilizer by screening the bacillus amyloliquefaciens strain W208, applies the microbial liquid bacterial fertilizer to a tea garden, and finds that the fertility effect is good by comparison; the soil of the tea garden can be obviously improved; the organic matter and humic acid content of the tea garden soil are obviously improved, and the improvement ranges are 81.7% and 73.9% respectively; the content of alkaline hydrolysis nitrogen, available phosphorus and quick-acting potassium in the tea garden soil can be obviously improved, and the improvement ranges are 81.4%, 53.0% and 30.5% respectively; the diversity of the tea garden soil microbial communities can be obviously improved; the method can obviously increase the yield of the tea, improve the quality of the tea, and obviously increase the contents of tea water extract, free amino acid and aroma substances, and particularly obviously reduce the content of caffeine in the tea by 24.3 percent. It is known that dry tea leaves contain a certain amount of caffeine. The high content of caffeine causes the tea to become bitter; in addition, caffeine can excite the central nervous system of people and enhance the excitation of cerebral cortex, and especially, insomnia is easy to cause by a large number of people drinking tea at night. The liquid bacterial manure provided by the invention can reduce caffeine in tea, remarkably improve the taste of tea and solve the problem of easy insomnia when drinking tea. In addition, the invention effectively solves the problems of short storage time and fast fertilizer efficiency attenuation of the liquid bacterial manure in the industry by adding the organic acid into the liquid bacterial manure. The preparation method is simple, low in cost and simple to use, is safe to people and livestock, does not cause environmental pollution, and has important significance for promoting tea production and sustainable development of ecological tea gardens.
Drawings
FIG. 1 shows the colony morphology of Bacillus amyloliquefaciens W208.
FIG. 2 is a scanning electron micrograph of Bacillus amyloliquefaciens W208.
FIG. 3 is a gram stain of Bacillus amyloliquefaciens W208.
FIG. 4 is a temperature optimum curve for growth of Bacillus amyloliquefaciens W208.
FIG. 5 is a pH optimum curve for growth of Bacillus amyloliquefaciens W208.
FIG. 6 is a graph showing the growth of Bacillus amyloliquefaciens W208.
Figure 7 is a graph of the structural differences in the effect of the invention and blank control on tea garden soil microbial communities.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. In the following examples, unless otherwise specified, the culture medium and experimental conditions in the examples were those of the conventional ones. The test materials, reagents and the like used in the examples are commercially available unless otherwise specified.
Example 1 acquisition and characterization of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) W208
1. Strain isolation
Soil samples were collected from farmlands containing rotten straw in shorea, countryside, shorea, jin' an, Fujian province. Weighing 5g of soil sample in a sterilized centrifuge tube, adding 5 times of volume of sterile water, filtering the soil, taking filtrate, inoculating the filtrate to an LB (lysogeny broth) plate culture medium, performing constant-temperature culture at 37 ℃ until bacterial colonies appear, selecting bacteria with good edge growth by using an inoculating needle, inoculating the bacteria on a new LB plate culture medium, after the newly-inoculated endophyte grows into bacteria, selecting the endophyte at the edge of the bacteria, performing streak culture, repeating the steps until a single bacterial colony is obtained, and naming the single bacterial colony as a bacterial strain W208. Wherein the LB plate culture medium comprises: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride and 15g/L of agar, wherein the pH value is 7.0, and the sterilization is carried out for 20min at 121 ℃.
2. Identification of strains
(1) And (3) observing colony and thallus morphology: the strain W208 is subjected to streak culture on an LB plate culture medium, and after the strain is cultured for 48 hours at the constant temperature of 37 ℃, the shape of a bacterial colony is raised, unsmooth, opaque, milky white and dendritic at the edge, and the morphology of the bacterial colony is shown in figure 1. Observed under a scanning electron microscope, the thallus is rod-shaped, has a length of 1.0-2.0 μm and a width of 0.3-0.6 μm, and has flagella, and a scanning electron microscope image thereof is shown in FIG. 2. Gram staining was performed and was positive, and the gram staining results are shown in fig. 3.
(2) Culture characteristic observation of strain W208: respectively inoculating the strain W208 into an LB liquid culture medium according to the inoculum size of 1.5 percent of the volume ratio, placing the strain in shaking culture at different temperatures for 24 hours, wherein the strain can grow in the environment of 15-45 ℃, the strain grows fastest in the environment of 30-40 ℃, and the optimal growth curve is shown in figure 4; and (3) placing the strain in different initial pH value environments for shake cultivation for 24 hours, wherein the strain can grow in the environment with the pH value ranging from 3 to 9, the strain grows fastest in the environment with the pH value ranging from 6 to 8, and the growth curve of the optimal pH value is shown in figure 5. Culturing in a shaker at 37 deg.C and pH 7.0, collecting culture solution at 0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, and 96h, measuring absorbance at 600nm wavelength, and drawing growth curve, as shown in FIG. 6, starting to enter logarithmic growth phase at 6h, reaching growth peak at 24h, entering stationary phase at 24h, and reaching 24 h. Wherein the LB liquid culture medium comprises: 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride, wherein the pH value is 6.0-8.0, and the sterilization is carried out for 20min at 121 ℃.
(3) Physiological and biochemical characteristics of strain W208: strain W208 is capable of growing in a medium containing glucose, mannose, xylose, fructose, sucrose, raffinose, gentiobiose, cellobiose, maltose, melibiose, lactose, stachyose, trehalose, sorbitol, mannitol, starch and gelatin as carbon sources; the results of a spore formation test, an oxidase test, a catalase test and a starch hydrolysis test are positive, the result of a cyst expansion test is negative, and the test is sensitive to rifamycin SV, lincomycin, lithium chloride, potassium tellurite, aztreonam, sodium butyrate, D-serine, sodium bromate, 1% sodium lactate, 1% sodium chloride, 4% sodium chloride and 8% sodium chloride, and is insensitive to oleandomycin acetate, vancomycin, minocycline, tetrazole blue, tetrazole violet, tetradecane sodium sulfate and nalidixic acid, and the results are shown in Table 1.
TABLE 1 physiological and biochemical characteristics of Strain W208
Figure BDA0001669820050000041
Figure BDA0001669820050000051
(4) Molecular biological characterization of strain W208: a bacterial whole genome rapid extraction kit is adopted to extract the whole genome of the strain W208, PCR is carried out by selecting bacterial 16S rDNA universal primers 27F and 1492R, and then sequencing analysis is carried out. And comparing sequencing results through BLAST, and identifying the strain as bacillus amyloliquefaciens, wherein the strain is named as: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) W208 and is preserved in the general microbiological center of China Committee for culture Collection of microorganisms at 2016, 9 and 13 days, with the preservation numbers: CGMCC No. 13006. The address of the depository: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101.
example 2 preparation of microbial liquid bacterial manure
(1) Preparation of LB medium: 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20 min.
Preparation of a fermentation medium: taking starch wastewater from a potato starch factory as a raw material, adding 10-30 g/L of soybean meal, adjusting the pH value to 6.0-8.0, and sterilizing at 121 ℃ for 20min to obtain the potato starch.
(2) Inoculating the bacillus amyloliquefaciens W208 obtained in the example 1 to an LB culture medium, and carrying out shaking culture for 24 hours at 180r/min in a constant temperature shaking table at 37 ℃ to prepare liquid seeds;
(3) inoculating liquid seeds into a fermentation culture medium in a fermentation tank according to the inoculation amount of 5% by mass volume, aerating, fermenting at 37 ℃ for 72 hours, supplementing the fermentation culture medium for the first time in 24 hours of fermentation, wherein the volume of the supplemented liquid is 50% of the volume of the fermentation liquid before supplementation, supplementing the fermentation culture medium for the second time in 56 hours of fermentation, the volume of the supplemented liquid is 30% of the volume of the fermentation liquid before supplementation, and detecting the bacillus amyloliquefaciens-containing spores in 72 hours of fermentationThe number of viable bacillus is 2.06X 108cfu/ml, and collecting fermentation liquor to obtain the microbial liquid bacterial manure.
Example 3 application of microbial liquid bacterial manure to tea production
The test is carried out in Hangzhou county flood pool tea field (with an elevation of about 1000 m) in Longyan City of Fujian province, the variety of the tea tree is Tieguanyin, and the tree age is 12 years. The planting mode is close planting in ridge planting, and the planting density is 3000-. The physical and chemical index parameters of the initial soil of the tea garden are as follows: the pH value is 5.8, the total nitrogen is 0.203%, the total phosphorus is 0.026%, the total potassium is 1.12%, the alkaline hydrolysis nitrogen is 138.1mg/kg, the available phosphorus is 106.7mg/kg, the quick-acting potassium is 297mg/kg, the organic matter is 45.0g/kg, and the humic acid is 17.1 g/kg.
The test was divided into two groups: blank Control (CK) and test (W208 YF).
Wherein the blank control group (CK) is applied with clear water at an application rate: 1500L/mu;
the test group (W208YF) applied the liquid bacterial manure of the microorganism prepared in example 2 of the present invention, which was diluted 50-fold with the fermentation medium before use, at the application rate: 1500L/mu.
Each treatment area is 0.5 mu, each treatment is repeated for 4 times, and the foliar spraying and furrow application are carried out in 2016, 8 months, 10 days and 9 months, 1 day, wherein the foliar spraying amount and the furrow application amount respectively account for about 50 percent. Soil and tea leaves are picked in 2016, 10 and 20 days, and the tea leaves are processed according to the Tie Guanyin green-making process to prepare dry tea.
And (4) detecting the microbial flora of the soil in the tea gardens and the yield and quality of the tea leaves treated differently.
1. The detection method comprises the following steps:
the tea water extract is determined according to GBT 8305 and 2013 tea water extract determination;
the total amount of free amino acids is determined according to GBT 8314 and 2013 total amount of free amino acids in tea;
the tea polyphenol determination refers to a method for detecting the contents of tea polyphenol and catechins in tea leaves in GBT 8313-2008;
the caffeine determination refers to GBT 8312 and 2013 Thea caffeine determination;
tea aroma component detection: (1) sample treatment: the aroma extraction method adopts HS-SPME: weighing 10.0g of the ground tea sample in a 150mL triangular flask, adding 25 μ L of 2mg/mL ethyl decanoate (internal standard) and 100mL boiling distilled water, covering tightly with a head space screw cap of a silica gel spacer, and stirring at a speed of: 450rpm, balancing in a 50 deg.C oven for 5min, inserting the extraction head into a triangular flask, adsorbing above the liquid surface of the tea soup for 40min at 50 deg.C, and desorbing at 230 deg.C for 5min at GC-MS sample inlet. (2) Gas chromatography-mass spectrometry conditions: a chromatographic column: HP-5MS (30m × 0.25mm ID × 0.25 μm film thickness). The carrier gas was high purity helium. Sample inlet temperature: 230 ℃ to 230 ℃. Pulse without shunting, sample introduction of 1 μ L, column flow rate: 1 mL/min. Interface temperature of chromatography-mass spectrometry: at 250 ℃ to obtain a mixture. Ion source temperature: 230 ℃ to 230 ℃. An ionization mode: EI. Electron energy: 70 eV. Temperature programming parameters: maintaining at 50 deg.C for 3min, increasing to 180 deg.C at 5 deg.C/min, maintaining for 3min, increasing to 230 deg.C at 10 deg.C/min, and maintaining for 5 min. (3) GC-MS analysis: the mass spectrum data obtained by GC-MS analysis is searched in NIST08, NIST27 and WILEY7 standard spectrum library by a computer, and is checked by combining with relevant documents to determine the chemical components, and the components are further determined by combining with the parameters such as retention time, mass spectrum, actual components and retention index. The relative contents of the components were quantitatively analyzed by peak area normalization (subtraction of solvent peak).
The tea garden soil fertility detection method comprises the following steps: the pH value, organic matter, alkaline hydrolysis nitrogen, available phosphorus, quick-acting potassium, total nitrogen, total phosphorus, total potassium and humic acid content are respectively detected according to NY/T1377 + 2007, NY/T1121.6-2006, soil agricultural chemical analysis method, NY/T1121.7-2006, NY/T889 + 2004, NY/T53-1987, NY/T88-1988, NY/T87-1988 and soil agricultural chemical analysis method. And (3) detecting microorganisms in the tea garden soil: according to the kit instructions (
Figure BDA0001669820050000073
SPIN Kit for soil, MPBIO corporation, usa) and then analyzing the diversity of soil microorganisms using 16S rDNA sequencing method and bioinformatic analysis software.
The detection results are as follows:
TABLE 2 yield and physicochemical indices of test (W208YF) and blank Control (CK) tea leaves
Figure BDA0001669820050000071
Note: results are expressed as mean ± standard deviation (n-4), and the upper right of the same row of data indicates significance compared to CK (P <0.05, independent sample t-test).
As can be seen from Table 2, compared with CK, the yield of W208YF tea is obviously increased (P <0.05), the amplification is 11.1%, and the microbial liquid bacterial manure prepared by the invention can obviously improve the yield of tea. Meanwhile, the detection result of physical and chemical indexes shows that the content of water extract and the content of free amino acid of the W208YF tea are both obviously increased (P <0.05), the increase of the water extract and the content of the free amino acid relative to CK are respectively 10.6% and 8.1%, the content of caffeine is obviously reduced (P <0.05), the reduction amplitude is 24.3%, the content of tea polyphenol is slightly increased, but the difference is not obvious (P > 0.05).
TABLE 3 aroma component content of tea leaves of test group (W208YF) and blank control group (CK)
Figure BDA0001669820050000072
Figure BDA0001669820050000081
Figure BDA0001669820050000091
Note: results are expressed as mean ± standard deviation (n-4), and the upper right of the same row of data indicates significance compared to CK (P <0.05, independent sample t-test).
The detection results in table 3 show that the application of the microbial liquid bacterial manure prepared by the invention can significantly improve the content of aromatic substances such as tea β -ocimene, nonanal, perillene, cis-3-hexenylbutyl ester, hexyl butyrate, trans-2-hexenylbutyl ester, 2-methyl-butanoic acid leaf alcohol ester, hexanoic acid leaf alcohol ester, nerolidol and hexanoestrol (P is less than 0.05), and significantly reduce the content of benzaldehyde, phenylacetaldehyde and indole (P is less than 0.05).
TABLE 4 physicochemical indices of soil in test group (W208YF) and blank control group (CK) tea garden
Figure BDA0001669820050000092
Note: results are expressed as mean ± standard deviation (n-4), and the upper right of the same row of data indicates significance compared to CK (P <0.05, independent sample t-test).
Table 4 shows that, compared with CK, the microbial liquid bacterial fertilizer provided by the present invention has significantly higher total nitrogen content, alkaline hydrolysis nitrogen, available phosphorus, available potassium, organic matter and humic acid content (P <0.05), and the increases thereof relative to CK group respectively reach 76.3%, 81.4%, 53.0%, 30.5%, 81.7% and 73.9%, which indicates that the application of the microbial liquid bacterial fertilizer W208YF of the present invention can significantly improve the utilization rate of the tea garden soil fertilizer and improve the soil fertility.
TABLE 5 microbial diversity of test (W208YF) and blank Control (CK) tea garden soils
Figure BDA0001669820050000101
Note: results are expressed as mean ± standard deviation (n-4), and the upper right of the same row data indicates significance compared to the blank control (P <0.05, independent sample t-test).
Table 5 shows that the indexes of Shannon and Simpson of the tea garden soil microbial community are significantly higher (P <0.05) by applying the liquid microbial fertilizer provided by the present invention, which indicates that the abundance and uniformity of the tea garden soil microbial community can be significantly improved by applying the liquid microbial fertilizer provided by the present invention.
The β diversity of the microbial community can be generally carried out by adopting a PCoA (Principal Coordinate Analysis) method, the difference of the microbial community of CK and W208YF (4 repeats in each group) tea garden soil is analyzed by the PCoA method, and the result is shown in figure 7. As can be seen from figure 7, the W208YF can be obviously distinguished from the CK, which shows that the microbial liquid bacterial manure provided by the invention can obviously regulate the microbial community structure of the tea garden soil.
The test results show that the microbial liquid bacterial fertilizer prepared by the method can obviously increase the soil fertility of tea gardens and the diversity of microbial communities, obviously improve the yield of tea leaves, obviously reduce caffeine in the tea leaves, and has obvious effects of improving the taste of the tea leaves and improving the quality of the tea leaves.
Example 4 storage stability of microbial liquid bacterial manure of the invention
Taking 4 parts of the microbial liquid bacterial manure prepared in the example 2, wherein each part is 1kg, and 3 parts of the microbial liquid bacterial manure are respectively added with acetic acid, the adding mass of the acetic acid is 0.01 percent, 0.05 percent and 0.1 percent of the mass of the microbial liquid bacterial manure, and the untreated part is a blank control.
4 parts of liquid bacterial manure are respectively packaged and stored at normal temperature (25 +/-1 ℃), and the survival number of cells is detected every month. The detection method comprises the following steps: plate counting method; calculating the survival rate of viable bacteria of the microbial liquid bacterial manure under different treatments according to the following formula:
the survival rate is the number of living bacteria of the microbial liquid fertilizer when the microbial liquid fertilizer is stored for N months/the number of the initial living bacteria of the microbial liquid fertilizer is multiplied by 100 percent, wherein N is 1, 2, 3 or 4.
TABLE 6 survival rates of liquid bacterial manure from different treated microorganisms
Figure BDA0001669820050000102
The detection result shows that the survival rate of viable bacteria of the microbial liquid bacterial fertilizer can be basically kept stable within 3 months when the microbial liquid bacterial fertilizer prepared by the invention is added with acetic acid, and the same technical effect can be achieved by replacing the acetic acid with formic acid and citric acid.
In addition, the microbial liquid bacterial manure added with the organic acid is diluted and applied by the same method after the pH is adjusted to 7.0 by using a proper amount of sodium hydroxide solution with the concentration of 6mol/L before use, and the technical effect is basically consistent with that of the embodiment 3.
The above are only a few specific embodiments of the present invention. The present invention is not limited to the above embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims (6)

1. The microbial liquid bacterial fertilizer is characterized by comprising 1.8 multiplied by 10 viable bacteria8cfu/ml~2.2×108cfu/mL of fermentation broth of Bacillus amyloliquefaciens added with organic acid, wherein the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens: (C)Bacillus amyloliquefaciens) W208, the strain is preserved in the China general microbiological culture Collection center on 2016, 9 and 13 days, and the preservation number is as follows: CGMCC No. 13006; the mass ratio of the organic acid to the fermentation liquor is 0.01-2.0: 100.
2. The microbial liquid bacterial fertilizer according to claim 1, wherein the organic acid is any one of formic acid, acetic acid or citric acid.
3. A preparation method of a microbial liquid bacterial fertilizer is characterized by comprising the following steps:
(a) inoculating the bacillus amyloliquefaciens strain into an LB culture medium, and carrying out shaking culture for 24 hours at 180r/min in a constant-temperature shaking table at 30-40 ℃ to prepare liquid seeds; the collection name of the bacillus amyloliquefaciens strain is bacillus amyloliquefaciens (B)Bacillus amyloliquefaciens) W208, deposited in the China general microbiological culture Collection center at 2016, 9, 13 and the preservation number is: CGMCC No. 13006;
(b) inoculating the liquid seeds into a fermentation culture medium according to the inoculation amount of 5% by mass volume, aerating, fermenting for 70-74 hours at 30-40 ℃, supplementing the fermentation culture medium in the fermentation process, wherein the viable count of the bacillus amyloliquefaciens in the liquid to be fermented is 1.8 multiplied by 108cfu/ml ~2.2×108And (3) collecting the fermentation liquor when cfu/mL is reached, and adding organic acid into the prepared fermentation liquor, wherein the adding mass of the organic acid is 0.01-2.0% of the mass of the fermentation liquor, so as to obtain the microbial liquid bacterial fertilizer.
4. The method for preparing a liquid microbial fertilizer as claimed in claim 3, wherein the organic acid is any one of formic acid, acetic acid and citric acid.
5. The preparation method of the microbial liquid bacterial fertilizer as claimed in claim 3 or 4, wherein the fermentation culture medium is a liquid culture medium prepared by taking starch wastewater from a potato starch factory as a raw material, adding 10-30 g/L of soybean meal powder, adjusting the pH value to 6.0-8.0 and sterilizing.
6. Use of a microbial liquid bacterial manure according to claim 1 or 2 in the production of tea.
CN201810498954.8A 2018-05-23 2018-05-23 Microbial liquid bacterial fertilizer and preparation method and application thereof Active CN108707560B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810498954.8A CN108707560B (en) 2018-05-23 2018-05-23 Microbial liquid bacterial fertilizer and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810498954.8A CN108707560B (en) 2018-05-23 2018-05-23 Microbial liquid bacterial fertilizer and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN108707560A CN108707560A (en) 2018-10-26
CN108707560B true CN108707560B (en) 2020-06-16

Family

ID=63868383

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810498954.8A Active CN108707560B (en) 2018-05-23 2018-05-23 Microbial liquid bacterial fertilizer and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN108707560B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112322542A (en) * 2020-11-17 2021-02-05 青岛普罗百世生物科技有限公司 Bacillus amyloliquefaciens and influence thereof on soil nutrient content and enzyme activity

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2013276965A1 (en) * 2013-12-24 2015-07-09 Sumitomo Chemical Company Limited Method of controlling pests
CN107018995A (en) * 2017-04-27 2017-08-08 四川省农业科学院植物保护研究所 A kind of method that use biocontrol bacterial strain improves organic tea yield

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2013276965A1 (en) * 2013-12-24 2015-07-09 Sumitomo Chemical Company Limited Method of controlling pests
CN107018995A (en) * 2017-04-27 2017-08-08 四川省农业科学院植物保护研究所 A kind of method that use biocontrol bacterial strain improves organic tea yield

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
解淀粉芽孢杆菌微生物菌剂对茶叶产量和品质的影响;周晨光 等;《中国农学通报》;20140115;第30卷(第2期);第253-257页 *

Also Published As

Publication number Publication date
CN108707560A (en) 2018-10-26

Similar Documents

Publication Publication Date Title
CN108676755B (en) Microbial liquid fertilizer containing bacillus and preparation method and application thereof
CN109370929B (en) Application of saccharomyces cerevisiae in brewing wine
CN106978350B (en) Aspergillus niger and application thereof in preparation of puerarin compounds
CN112410244B (en) Bacillus aryabhattai and application thereof
CN107136122B (en) Biocontrol microbial inoculum for preventing and treating potato late blight
CN110358710B (en) Bacillus laterosporus and application thereof in preparation of disease-resistant and salt and alkali resistant functional microbial preparation
CN103627662A (en) Peanut bradyrhizobium sp. and application thereof
CN112852664B (en) Saccharomyces cerevisiae and method for improving yield of gamma-aminobutyric acid produced by saccharomyces cerevisiae
CN117736944A (en) Streptomyces griseus as well as microbial inoculum and application thereof
CN108707560B (en) Microbial liquid bacterial fertilizer and preparation method and application thereof
CN102433272A (en) Xanthobacter flavus DT8 and the use thereof for degrading cyclic ethers
CN102816717B (en) Staphylococcus cohnii and method for preparing 5-aminolevulinic acid by using staphylococcus cohnii
CN110791459B (en) Bacillus subtilis for preventing and controlling continuous cropping lily soil-borne blight and application thereof
CN110951625B (en) Penicillium phosphate solubilizing PSF and application thereof
CN117106614B (en) Rhizosphere bacterium pseudomonas solanacearum YIM B08402, microbial agent and application thereof
CN110272841B (en) Brevundimonas flavus and application thereof in codonopsis pilosula
CN114703069B (en) Epicoccus nigrum fermentation product, preparation method and application thereof
CN115651846A (en) Trichoderma fungicide, biological organic fertilizer, preparation method and application thereof
CN112280714B (en) Pseudomonas aeruginosa 8-7 and application thereof
CN114196583A (en) Thermophilic carbon monoxide chain mould, synergistic biological agent containing same and application
CN114921362A (en) Camellia oleifera endophytic actinomycetes with insoluble phosphorus dissolving and growth promoting functions and application thereof
CN113862179A (en) Rhodopseudomonas palustris, application and method for preparing 5-ALA by using rhodopseudomonas palustris
CN111411046A (en) Dark color endophytic fungus agent and application thereof
CN102433275B (en) Ochrobactrum and application thereof
CN115786166B (en) Klebsiella pneumoniae and application thereof

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
CP03 Change of name, title or address
CP03 Change of name, title or address

Address after: 350003 No. 54, 247 North Road, Gulou District, Fujian, Fuzhou

Patentee after: Fujian Academy of Agricultural Sciences Agricultural Product Processing Research Institute

Address before: 350000 No. 54 North 247 Road, Fujian, Fuzhou

Patentee before: INSTITUTE OF AGRICULTURAL ENGINEERING TECHNOLOGY, FUJIAN ACADEMY OF AGRICULTURAL SCIENCES