CN112205407A - Agricultural bacteriostatic agent from microorganisms and preparation method thereof - Google Patents

Agricultural bacteriostatic agent from microorganisms and preparation method thereof Download PDF

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CN112205407A
CN112205407A CN202011051322.0A CN202011051322A CN112205407A CN 112205407 A CN112205407 A CN 112205407A CN 202011051322 A CN202011051322 A CN 202011051322A CN 112205407 A CN112205407 A CN 112205407A
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bacteriostatic agent
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赵峰
董梅
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Qufu Normal University
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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
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides

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Abstract

The invention relates to the field of prevention and control of agricultural pathogenic bacteria, in particular to an agricultural bacteriostatic agent derived from microorganisms and a preparation method thereof. The active component of the agricultural bacteriostatic agent is monorhamnolipid produced by microbial fermentation, and the concentration of the monorhamnolipid in the agricultural bacteriostatic agent is 0.01-0.5 wt%. The invention also provides a preparation method of the agricultural bacteriostatic agent, which comprises the following steps: 1) fermenting strains of a single rhamnolipid producing strain or a conventional rhamnolipid producing strain, 2) pretreating fermentation liquor of the single rhamnolipid producing strain, or separating a single rhamnolipid component from a rhamnolipid fermentation liquor extract, 3) diluting the fermentation liquor of the single rhamnolipid producing strain, or preparing a single rhamnolipid solution to prepare the bacteriostatic agent. The agricultural bacteriostatic agent provided by the invention is of microbial source, good in bacteriostatic effect and wide in bacteriostatic spectrum, and can be applied to prevention and treatment of agricultural diseases caused by plant pathogenic bacteria.

Description

Agricultural bacteriostatic agent from microorganisms and preparation method thereof
Technical Field
The invention relates to the field of agricultural biocontrol, in particular to an agricultural bacteriostatic agent derived from microorganisms and a preparation method thereof.
Background
The plant pathogenic bacteria can cause crops to have wilting, rotting, blotch, white leaves, epidemic diseases, blight and the like, and even cause plants to die. The plant pathogenic bacteria threaten the crop yield and influence the quality safety of agricultural products. Plant diseases caused by plant pathogenic bacteria bring huge economic loss to agriculture, so that the prevention and control of the agricultural pathogenic bacteria have important theoretical research and practical application values. The appearance of the chemical bacteriostatic agent brings an effective prevention and treatment approach for the harm of agricultural pathogenic bacteria. The chemical bacteriostatic agent has the defects of narrow bacteriostatic spectrum, easy generation of drug resistance of plant pathogenic bacteria and the like, and the chemical bacteriostatic agent, particularly some organic bacteriostatic agents, are not completely degraded to cause residues in the environment.
With the development of agriculture and the technological progress, the agricultural field is gradually inclined to develop and apply the bacteriostatic agent with higher efficiency, greenness, low toxicity and low residue. The biosurfactant is a metabolite which is synthesized by microorganisms and has an amphiphilic molecular structure, comprises glycolipids, lipopeptides and the like, and has the functions of resisting bacteria, reducing surface/interface tension, emulsifying, permeating and the like. The biosurfactant has a wide antibacterial spectrum on plant pathogenic bacteria, and can also enhance the defense capability of plants on bacteria and fungi. The biosurfactant is used for preventing and treating the agricultural pathogenic bacteria, is safe, effective, economical and environment-friendly, and accords with the strategy principle of prevention as the main and comprehensive prevention in agricultural prevention and treatment.
Disclosure of Invention
The invention aims to provide an agricultural bacteriostatic agent derived from microorganisms and a preparation method thereof, which are beneficial to solving the problems that a chemical bacteriostatic agent is narrow in bacteriostatic spectrum, easy to cause drug resistance of plant pathogenic bacteria, incomplete in degradation, easy to remain in the environment and the like in the control of agricultural pathogenic bacteria.
In order to achieve the purpose, the invention adopts the technical scheme that:
an agricultural bacteriostatic agent derived from microorganisms, wherein the active ingredient of the bacteriostatic agent is monorhamnolipid, and the concentration of the monorhamnolipid in the agricultural bacteriostatic agent is 0.01-0.5 wt%.
The concentration of the single rhamnolipid in the agricultural bacteriostatic agent is 0.02-0.2 wt%.
The agricultural bacteriostatic agent of microorganism source for the solution form, be the aqueous solution that contains single rhamnolipid, perhaps single rhamnolipid produces fermented culture, fermentation culture suspension or the fermentation supernatant that the fungus fermented gained contains single rhamnolipid, perhaps is with the diluent of above liquid.
The single rhamnolipid is a single rhamnolipid separated from a rhamnolipid mixture, or a single rhamnolipid extract of a fermentation culture obtained by fermenting a starting strain of a single rhamnolipid producing strain.
The preparation method of the agricultural bacteriostatic agent derived from the microorganisms comprises the following specific steps:
1) fermenting strains of the rhamnolipid producing bacteria or the conventional rhamnolipid producing bacteria for 3-6 days at the fermentation temperature of 30-40 ℃;
2) centrifuging 5000-10000 g of fermentation liquor of the rhamnolipid producing strain, removing thalli, insoluble substances and the like, and obtaining fermentation supernatant; or extracting a mixture of mono-rhamnolipid and di-rhamnolipid from conventional rhamnolipid producing strain fermentation liquor, and separating a mono-rhamnolipid component by using a thin layer chromatography or preparative HPLC (high performance liquid chromatography) method;
3) diluting the fermentation supernatant of the monorhamnolipid producing bacteria with clear water according to the concentration of the monorhamnolipid in the bacteriostatic agent to be prepared; or preparing a mono rhamnolipid solution by using clear water and the separated mono rhamnolipid.
The conventional rhamnolipid-producing bacteria refer to certain bacteria of the genus pseudomonas aeruginosa or klebsiella, all of which are capable of producing a rhamnolipid mixture comprising mono and di rhamnolipids.
The rhamnolipid producing strain is a strain obtained by genetically modifying a conventional rhamnolipid producing strain in a conventional rhamnolipid producing strainrhlABThe gene is a key gene for synthesizing the single rhamnolipid, andrhlCthe gene is a key gene for synthesizing the dirhamnolipid and a key gene for synthesizing the monorhamnolipidrhlABHeterologous expression construction into a suitable microbial hostIn a group of bacteria, or in a knockout of conventional rhamnolipid-producing bacteriarhlCGene construction knockout strains, and two kinds of genetic engineering bacteria can only express genes related to the synthesis of the single rhamnolipidrhlABSo that only mono rhamnolipids are produced.
The agricultural bacteriostatic agent derived from the microorganisms is applied to prevention and control of agricultural pathogenic bacteria.
The bacteriostatic agent can effectively inhibit the growth of plant pathogenic bacteria and pathogenic fungi, has broad-spectrum bacteriostatic action, and can be used for preventing and treating agricultural diseases caused by plant pathogenic bacteria.
The invention has the beneficial effects that:
the agricultural bacteriostatic agent is derived from the biosurfactant produced by microbial fermentation, and has the advantages of good bacteriostatic effect, wide bacteriostatic spectrum, low toxicity and ecological friendliness; the active component of the agricultural bacteriostatic agent is the monorhamnolipid, and compared with the dirhamnolipid, the hydrophilic end of the monorhamnolipid only contains one rhamnose ring, so the monorhamnolipid has better lipophilic property, and shows stronger cell-dissolving activity under the action of plant pathogenic bacteria, thereby causing the death of pathogenic bacteria cells. Therefore, the agricultural bacteriostatic agent derived from microorganisms is an efficient and green plant pathogenic bacteria bacteriostatic agent and can be applied to prevention and treatment of agricultural diseases caused by plant pathogenic bacteria.
Drawings
FIG. 1 is a bacteriostatic photograph of different rhamnolipids.
Detailed Description
The present invention is described in detail with reference to the following specific examples to provide a more complete understanding of the present invention to those of ordinary skill in the art, but the present invention is not limited thereto in any way. In the following examples, materials and reagents used were all available from Biochemical materials, Inc., unless otherwise specified.
Example 1: extracting and separating monorhamnolipid component from conventional rhamnolipid producing strain fermentation liquor
The rhamnolipid synthesized by conventional rhamnolipid producing bacteria is a mixture of mono and di rhamnolipids. In the examples for producing monorhamnolipids and dirhamnolipidsPseudomonas aeruginosa (Pseudomonas aeruginosa) SG（Zhao et al. Comparative studies on the structural composition, surface/interface activity and application potential of rhamnolipids produced by Pseudomonas aeruginosausing hydrolytic or hydrolytic substrates, bioreource Technology, 2020, 295, 122269) as the starting strain.
LB culture medium: 10g of peptone, 5g of yeast powder and 10g of NaCl, and adding deionized water to 1L, wherein the pH value is 7.0.
Fermentation medium: glycerol 45g, NaNO3 3.5g，K2HPO4∙3H2O 4.0g，KH2PO4 3.0g，MgSO4∙7H2O1.0 g, deionized water to 1L, pH 7.0.
Respectively weighing the reagents by using an electronic balance, and preparing corresponding culture media; subpackaging, sterilizing at 121 deg.C for 20 min.
After the culture medium is cooled to 20-40 ℃, a strain slant of the strain SG is scraped by an inoculating loop, inoculated into a triangular flask containing 100ml of LB culture medium, and cultured for 6-10 hours at 37 ℃ and 180 rpm/min until OD is reached600The value is 0.6-1.0, and strain SG seed liquid is obtained.
Then, strain SG seed liquid is inoculated into a fermentation medium in an inoculation amount (v/v) of 3%, and the strain SG seed liquid is cultured for 5 days under the conditions of 37 ℃ and 180 rpm and is used for fermenting and producing rhamnolipid, so that fermentation liquid containing the rhamnolipid and the dirhamnolipid is obtained.
Centrifuging the fermentation liquid at 8000g to remove insoluble substances such as thallus, extracting rhamnolipid product with chloroform/methanol (v/v, 2: 1) from supernatant, and vacuum rotary evaporating the extractive solution at 50 deg.C to obtain mixture of monorhamnolipid and birrhamnolipid.
The extracted rhamnolipid product is subjected to silica gel column chromatography, and the monorhamnolipid in the product is separated, wherein the separation method refers to related documents (Zhonghua. 2008. study on the bacterial adsorption of rhamnolipid and the modification effect of rhamnolipid on the bacterial surface).
The separated monorhamnolipid was verified by thin layer chromatography. A small amount of the separated monorhamnolipid is dissolved in dichloromethane, a 200mg/L monorhamnolipid sample is prepared, the sample is spotted on a silica gel plate, developed in a developing solvent of chloroform/methanol/acetic acid =65:15:2 (v/v/v), developed by a phenol-sulfuric acid developer, and the Rf value is calculated. The monorhamnolipid has only one point on a thin-layer silica gel plate, and the specific migration value (Rf value) of the monorhamnolipid is 0.85. If the resulting product is a mixture of mono and bis rhamnolipids, two points with different Rf values appear on the lamella plates. The Rf value of the monorhamnolipid is about 0.89, and the Rf value of the dirhamnolipid is about 0.55 (the relevant records of the thin-layer chromatography analysis of the monorhamnolipid and the dirhamnolipid are shown in Zhonghua-2008. the thallus adsorption of the rhamnolipid and the modification effect research of the rhamnolipid on the surface of the thallus). Therefore, it was demonstrated that the rhamnolipid component extracted and separated from the fermentation broth of the conventional rhamnolipid-producing bacterium is a mono rhamnolipid.
And (3) dissolving the separated monorhamnolipid with water to ensure that the concentration reaches 200-2000 mg/L, and using the monorhamnolipid as a plant pathogenic bacteria bacteriostatic agent.
Example 2: preparation of Monorhamnolipid fermentation broth
Genetic engineering bacterium pseudomonas stutzeri for selecting rhamnolipid product as completely monorhamnolipidPseudomonas stutzeri Rhl（Zhao et al. Heterologous production of Pseudomonas aeruginosarhamnolipid under and anaerobic conditions for microbial enhanced oil recovery. Journal of Applied Microbiology 2015, 118: 379-389) as starting strain for producing monorhamnolipids.
Using the LB medium and the fermentation medium of example 1, the slant 1 loop of the strain Rhl was inoculated into a flask containing 100ml of LB medium, and cultured at 30 ℃ and 180 rpm/min for 6 to 10 hours to OD600The value is 0.6-1.0, and strain Rhl seed liquid is obtained. Then, the fermentation medium was inoculated at an inoculum size of 3% (v/v) and cultured at 37 ℃ and 180 rpm for 5 days for fermentative production of monorhamnolipid to obtain a monorhamnolipid fermentation broth.
Diluting the obtained rhamnolipid fermentation liquor with water to ensure that the concentration of the rhamnolipid is 200-2000 mg/L, and using the rhamnolipid fermentation liquor as a bacteriostatic agent for plant pathogenic bacteria.
Example 3: extraction of monorhamnolipid from fermentation liquid of monorhamnolipid producing strain
The fermentation broth after completion of the culture in example 2 was centrifuged at 8000g to remove insoluble materials such as cells, and the monorhamnolipid in the fermentation broth was extracted by chloroform/methanol (v/v, 2: 1) extraction in example 1.
The extracted monorhamnolipid product was qualitatively verified by thin layer chromatography as in example 1.
The extracted monorhamnolipid is used for preparing an aqueous solution, wherein the concentration of the monorhamnolipid is 200-2000 mg/L, and the monorhamnolipid is used as a bacteriostatic agent for plant pathogenic bacteria.
Example 4: application example
The bacteriostatic agent obtained in the embodiment 1-3 is used for acting on bacteria and fungi, measuring the size of a bacteriostatic zone and evaluating the bacteriostatic effect.
Preparing LB liquid culture medium and potato glucose liquid culture medium, LB agar culture medium plate and PDA culture medium (potato glucose agar culture medium) plate, which are respectively used for culturing bacteria and fungi. Firstly, preparing seed liquid of bacteria and fungi to be tested by using liquid culture medium, and diluting the seed liquid by 105After doubling, respectively coating on corresponding agar plates; then, a sterile filter paper sheet with the diameter of 10mm is placed on the flat plate, 10 mu L of the bacteriostatic agent obtained in the embodiment 1-3 is respectively dripped on the filter paper sheet, and the concentration of the rhamnolipid is 500 mg/L. The plate was placed in an incubator at 30 ℃ for 2 days. After the culture was completed, the size (mm) of the zone of inhibition around the filter paper sheet was measured. The tested strains are escherichia coli (Ec), staphylococcus aureus (Sa), some plant pathogenic bacteria (Pa) screened in a laboratory which are not identified yet, some plant pathogenic fungus 1 (Pf 1) and some plant pathogenic fungus 2 (Pf 2), and the experimental results are shown in table 1.
Table 1: bacteriostatic results of three bacteriostatic agents
Figure 292294DEST_PATH_IMAGE002
Example 5: comparative example
The rhamnolipid product synthesized by conventional rhamnolipid producing bacteria is a mixture of mono and bis rhamnolipids. This example compares the bacteriostatic effects of mono and ordinary rhamnolipids (a mixture of mono and di rhamnolipids).
And (3) fermenting by using the pseudomonas aeruginosa SG which can simultaneously produce the monorhamnolipid and the dirhamnolipid in the example 1 as an initial strain to obtain fermentation liquor containing the monorhamnolipid and the dirhamnolipid.
The rhamnolipid product (mixture of mono and bis rhamnolipids) in the fermentation broth was extracted using chloroform/methanol (v/v, 2: 1) extraction as in example 1.
The monorhamnolipid component and the dirhamnolipid component were separated by silica gel column chromatography in example 1.
The separated monorhamnolipid is dissolved by distilled water to prepare an aqueous solution, the concentration reaches 500mg/L, and the aqueous solution is used as a bacteriostatic agent R1.
The separated dirhamnolipid is dissolved by distilled water to prepare an aqueous solution, the concentration reaches 500mg/L, and the aqueous solution is used as a bacteriostatic agent R2.
Referring to the bacteriostasis experiment method in example 4, bacteriostasis agents R1 and R2 were used to act on three plant pathogenic bacteria, and the bacteriostasis effects were compared. The test strains were a laboratory selection of a certain plant pathogenic bacterium (Pa), a certain plant pathogenic fungus 1 (Pf 1) and a certain plant pathogenic fungus 2 (Pf 2) which had not been identified, and the results of the experiments are shown in Table 2.
Table 2: bacteriostasis results of bacteriostat R1 and bacteriostat R2
Figure 412696DEST_PATH_IMAGE004
As shown in figure 1, the test strain is a certain plant pathogenic fungus 2 which is screened in a laboratory and is not identified, a bacteriostat R1 is prepared into an aqueous solution by separating monorhamnolipid with the concentration of 500mg/L, and a bacteriostat R2 is prepared into an aqueous solution by separating dirhamnolipid with the concentration of 500 mg/L. The bacteriostatic effects of R1 and R2 can be compared from the size of the bacteriostatic circle around the filter paper sheet in the figure, wherein the size of the bacteriostatic circle of R1 is 32mm, and the size of the bacteriostatic circle of R2 is 19 mm.
The bacteriostatic agent derived from microorganisms takes the rhamnolipid as an active ingredient, has good bacteriostatic effect, simple and convenient preparation, environmental protection and broad-spectrum bacteriostatic action, can be applied to the control of agricultural diseases caused by plant pathogenic bacteria, and has important popularization and application values in the field of agricultural biocontrol.

Claims (10)

1. An agricultural bacteriostatic agent derived from microorganisms, which is characterized in that: the active component of the bacteriostatic agent is monorhamnolipid.
2. The agricultural bacteriostatic agent of claim 1, which is characterized in that: the concentration of the monorhamnolipid in the bacteriostatic agent is 0.01-0.5 wt%.
3. The agricultural bacteriostatic agent of claim 1, which is characterized in that: the concentration of the monorhamnolipid is 0.02wt% -0.2 wt%.
4. The microbial-derived agricultural bacteriostatic agent of claims 1 to 3, which is characterized in that: the bacteriostatic agent is an aqueous solution containing the monorhamnolipid, or a fermentation culture, a fermentation culture suspension or a fermentation supernatant which are obtained by fermenting the monorhamnolipid producing bacteria and contain the monorhamnolipid, or a diluent of the liquid.
5. The microbially-derived agricultural bacteriostatic agent of claim 4, wherein: the single rhamnolipid is a single rhamnolipid separated from a rhamnolipid mixture, or a single rhamnolipid extract of a fermentation culture obtained by fermenting a starting strain of a single rhamnolipid producing strain.
6. The method of preparing the microbial agricultural bacteriostatic agent of claims 1 to 3, comprising: 1) fermenting strains of a single rhamnolipid producing strain or a conventional rhamnolipid producing strain, 2) pretreating fermentation liquor of the single rhamnolipid producing strain, or separating a single rhamnolipid component from a rhamnolipid fermentation liquor extract, 3) diluting the fermentation liquor of the single rhamnolipid producing strain, or preparing a single rhamnolipid solution to prepare the bacteriostatic agent.
7. The method of preparing an agricultural bacteriostatic agent of microbial origin according to claim 6, wherein the step 1): fermenting and culturing the rhamnolipid producing strain or the conventional rhamnolipid producing strain for 3-6 days at the fermentation temperature of 30-40 ℃.
8. The method of preparing an agricultural bacteriostatic agent of microbial origin according to claim 6, wherein the step 2): centrifuging 5000-10000 g of fermentation liquor of the rhamnolipid producing strain, removing thalli and insoluble substances, and obtaining fermentation supernatant; or extracting a mixture of the monorhamnolipid and the dirhamnolipid from fermentation liquor of conventional rhamnolipid producing bacteria, and separating a monorhamnolipid component by using a thin layer chromatography or a preparative HPLC method.
9. The method of preparing an agricultural bacteriostatic agent of microbial origin according to claim 6, wherein the step 3): diluting the fermentation supernatant of the monorhamnolipid producing bacteria with clear water according to the concentration of the monorhamnolipid in the bacteriostatic agent to be prepared; or preparing a mono rhamnolipid solution by using clear water and the separated mono rhamnolipid.
10. The use of the microbial-derived agricultural bacteriostatic agent of claims 1 to 3, wherein: the bacteriostatic agent is applied to prevention and treatment of agricultural pathogenic bacteria.
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP4088576A1 (en) * 2021-05-14 2022-11-16 Rheinische Friedrich-Wilhelms-Universität Bonn Method of pathogen control

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RUYI SHA 等: "Producing cell-free culture broth of rhamnolipids as a cost-effective fungicide against plant pathogens", 《JOURNAL OF BASIC MICROBIOLOGY》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4088576A1 (en) * 2021-05-14 2022-11-16 Rheinische Friedrich-Wilhelms-Universität Bonn Method of pathogen control
WO2022238567A1 (en) * 2021-05-14 2022-11-17 Rheinische Friedrich-Wilhelms-Universität Bonn Method of pathogen control

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