WO2021068556A1 - Rice blast fungicide prepared using lysinibacillus boronitolerans as chassis cell - Google Patents

Rice blast fungicide prepared using lysinibacillus boronitolerans as chassis cell Download PDF

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WO2021068556A1
WO2021068556A1 PCT/CN2020/097735 CN2020097735W WO2021068556A1 WO 2021068556 A1 WO2021068556 A1 WO 2021068556A1 CN 2020097735 W CN2020097735 W CN 2020097735W WO 2021068556 A1 WO2021068556 A1 WO 2021068556A1
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boron
resistant
medium
lysine
bacillus
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French (fr)
Chinese (zh)
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刘柱
晏承梁
马香
唐燕琼
李宏
高玉晓
林敏�
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海南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • 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

Definitions

  • the invention relates to a synthetic biology modification and application of biocontrol bacteria, in particular to a rice blast fungicide.
  • Rice blast is one of the important diseases of rice, which can cause a substantial reduction in rice production, and in severe cases, it can lead to a reduction of 40% to 50%, or even no grain harvest.
  • Rice blast is caused by the parasitic fungus (such as rice blast fungus), and the spread of the disease is spread by conidia in the air.
  • the optimum temperature for the development of the pathogen is 25°C to 28°C. High humidity is good for conidia formation and germination, while high humidity for more than one day and night is good for the spread of the disease.
  • Magnaporthe grisea belongs to the subphylum Deuteromycotina. In addition to infecting rice and causing it to infect rice blast, it can also cause skin diseases in humans and some animals.
  • Drug control use antibacterial chemicals to soak seeds in advance, spray pesticides, etc.
  • chemical agents has brought many conveniences and effects to the prevention and control of plant diseases, the production cost of chemical agents is inherently high, and after the use of chemical agents for sterilization, environmental pollution, excessive pesticide residues in agricultural products and pathogen resistance will follow. Negative effects such as the formation of AIDS have aroused widespread concern in the society.
  • the present invention proposes a boron-resistant lysine bacillus (Lysinibacillus boronitolerans), which was deposited in the China Center for Type Culture Collection on October 8, 2019, referred to as CCTCC, and the deposit number is CCTCC NO.M 2019773.
  • CCTCC boron-resistant lysine bacillus
  • a bactericide includes the above-mentioned boron-resistant Lysine Bacillus fermentation broth (Lysinibacillus boronitolerans) and auxiliary materials.
  • the auxiliary material is one or more of water, liquid culture medium, solid culture medium, and glycerin.
  • the above bactericide wherein the fermentation broth of Boron-resistant Lysine Bacillus is the above-mentioned Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) cultured for 24-96 hours, and the OD 600 is 4.8-2.5 hours Of fermentation broth.
  • Lysinibacillus boronitolerans Lysinibacillus boronitolerans
  • a boron-lysine-resistant bacillus as described above is used as a chassis cell-based transformation module.
  • a method for preventing or treating rice blast includes: obtaining the above-mentioned fungicide; and mixing the fungicide with a medium.
  • the medium is a vector carrying Magnaporthe oryzea.
  • the carrier is one or more of a seed, a plant, the soil where the plant grows, and a medium for cultivating the plant.
  • the bactericide as described above wherein the fermentation broth of Lysinibacillus boronitolerans is the fermentation broth of Lysinibacillus boronitolerans with 200ml broth (initial OD is 0.02OD 600 /ml)/500ml LB medium
  • the fermentation product is obtained when the OD value of the bacteria liquid reaches 4.8, 4.0, 2.5, and the OD value of the bacteria liquid reaches 4.8, 4.0, 2.5, and the culture is carried out at 150r/min at 37°C for 24h, 60h or 96h.
  • Rice blast is an important disease of rice, causing serious economic losses to agricultural production.
  • the extensive use of chemical pesticides also poses a huge threat to the environment and food.
  • Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) can not only reduce the occurrence and damage of rice blast to a large extent, but also has no impact on the ecological environment. It is expected to be a high-quality chassis microorganism for synthetic biology research and bring to the control of rice blast. New direction.
  • Fig. 1 is the identification of potential biocontrol bacteria isolated from soil according to an embodiment of the present application.
  • Figure 1A is a gel electrophoresis separation map of bacterial genomic DNA separated and purified from soil
  • Figure 1B is a gel electrophoresis separation map of PCR products obtained by specific amplification of genomic DNA with 16S primers
  • Figure 1C is a through The phylogenetic tree of potential biocontrol bacteria obtained after sequence alignment, through sequence comparison analysis, identified the obtained potential biocontrol bacteria as Lysinibacillus boronitolerans (Lysinibacillus boronitolerans);
  • FIG. 2 shows the growth inhibitory effect of the fermentation broth of boron-lysine-resistant Bacillus at different stages on the rice blast fungus according to an embodiment of the present application.
  • Figures 2A, 2B, and 2C are the experimental groups, in which the sterile fermentation filtrate obtained after fermentation with boron-resistant lysine Bacillus for 24h, 60h, and 96h was added to the culture medium;
  • Figure 2D is the control group. Add sterile fermentation filtrate of Boron-Lysine-resistant Bacillus to the medium;
  • Fig. 3 shows the antibacterial activity detection of different concentrations of ethyl acetate extracts of 96h fermentation broth of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) against Magnaporthe oryzea according to an embodiment of the present application.
  • the concentration of the crude extract is 1mg/ml, 5mg/ml, 10mg/ml and 15mg/ml;
  • Figure 4 shows the detection of antibacterial activity of extracts obtained by precipitation of ammonium sulfate with different saturations in 96h fermentation broth of Lysinibacillus boronitolerans according to an embodiment of the present application against Magnaporthe oryzea .
  • the concentration is a concentration within a certain numerical range.
  • concentration is expressed by the OD value
  • OD 600 of 4.8 appears in the application
  • the actual OD value is about 4.8, such as the OD 600 value is 4.8 ⁇ 0.6.
  • the time required to ferment and cultivate the bacteria to a certain level is a certain time that cannot be accurate to minutes and seconds, and the required time is related to the environmental temperature of bacterial fermentation, culture, and the type of culture medium, so it appears in this application
  • the time related to bacterial fermentation, cultivation, growth, etc. are approximate times, not definite times.
  • the fermentation broth mentioned in this application refers to the fermentation product-containing liquid produced by bacteria after fermentation.
  • the fermentation broth of Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) is a 200ml broth (initial OD). About 0.02OD 600 /ml)/500ml LB medium filling volume, the fermentation product obtained by culturing at 37°C at 150r/min for about 24h, 60h or 96h.
  • the fermentation time of boron-resistant lysine bacillus was about 24, 60h and 96h
  • the OD value of the bacterial solution was about 4.8, 4.0 and 2.5 (OD value decreased with the extension of fermentation time).
  • the precipitate is removed by centrifugation, the fermentation supernatant is collected, and then filtered with a 0.22 ⁇ m filter membrane to obtain a sterile fermentation filtrate.
  • the toxic medium method mentioned in this application is also called the growth rate method, which is one of the conventional methods for the determination of the virulence of fungicides, and is suitable for fungi that do not grow spores but have faster hyphae growth.
  • the virulence of the agent can be measured by the growth rate of the colony.
  • Toxic medium method is to mix the test agent with the culture medium, and measure the virulence of the medicine by the growth rate of the colony on the culture medium.
  • the colony growth rate is generally expressed by the time (days or hours) required for the colony to reach a given size, or the size of the colony diameter per unit time.
  • the perforating method mentioned in this application refers to perforating a test plate with a sterilized perforator or steel pipe, and then injecting a certain amount of the sample to be tested into the hole, and measuring the inhibition zone after incubating for a period of time. A way of size.
  • the extraction method mentioned herein refers to the use of the difference in solubility or partition coefficient of a compound in two immiscible (or slightly soluble) solvents to transfer the compound from one solvent to another. After repeated extractions, most of the compounds are extracted.
  • Ethyl acetate is a medium polar solvent, which can extract a variety of substances from the fermentation filtrate of bacteria, such as molecules with less polarity (such as glucose or glycosides), molecules with very small polarity (such as certain Paraffin), molecules containing salt structures (such as amino acids), etc.
  • the ammonium sulfate precipitation mentioned in this application refers to the technique of using different concentrations of ammonium sulfate solutions to precipitate and separate proteins. Commonly used to separate immunoglobulins.
  • the ammonium sulfate precipitation method can be used to concentrate and partially purify proteins from a large number of crude preparations.
  • the high concentration of salt ions in the protein solution can compete with the protein for water molecules, thereby destroying the hydration film on the surface of the protein, reducing its solubility, and making it precipitate out of the solution.
  • the solubility of various proteins is different, so different concentrations of salt solutions can be used to precipitate different proteins. This method is called salting out. Salt concentration is usually expressed in terms of saturation.
  • Ammonium sulfate is the most widely used because of its high solubility, low temperature coefficient and difficulty in denaturing protein.
  • 16S rDNA is a DNA sequence corresponding to 16S rRNA on bacterial chromosomes, which is present in all bacterial chromosomal genes. Its internal structure is composed of conserved regions and variable regions. The variable regions within the molecule show the specificity of bacteria at the level of different classification levels.
  • ribosomal RNA in bacteria There are three types of ribosomal RNA in bacteria, namely 5S, 16S and 23S rRNA.
  • 5S rRNA is easy to analyze, but the number of nucleotides is too small, consisting of only a few dozen nucleotides, resulting in insufficient genetic information and cannot be used for classification research; 23S rRNA is too large for its molecular weight and contains nucleosides.
  • the amount of acid is almost twice that of 16S rRNA, and it is difficult to analyze and not select it for classification research.
  • 16S rRNA is usually used for bacterial classification research.
  • 16S rRNA is ubiquitous in prokaryotes (the homologous molecule in eukaryotes is 18S rRNA). rRNA participates in the process of biological protein synthesis, its function is indispensable to any organism, and it remains unchanged during the long course of biological evolution, which can be regarded as the time clock of biological evolution.
  • 16S rRNA molecules contain both highly conserved sequence regions and moderately conserved and highly variable sequence regions. Therefore, it is suitable for the study of various biological relationships with different evolutionary distances.
  • the relative molecular weight of 16S rRNA is moderate, about 2kb nucleotides, which is convenient for sequence analysis. Therefore, it can be used as a good tool to measure the evolution and genetic relationship of various organisms.
  • the coding gene of 16S rRNA is 16S rDNA. It is difficult to directly extract 16S rRNA from bacteria, and the extracted RNA is easily degraded and difficult to store. Therefore, 16S rDNA is usually used to identify the type of bacteria.
  • PCR or polymerase chain reaction mentioned in this application, means that under the catalysis of DNA polymerase, the parent strand DNA is used as a template, and a specific primer is used as the starting point for extension. Through the steps of denaturation, annealing, extension, etc., it replicates with the parent strand in vitro.
  • PCR is an in vitro synthetic amplification technology that can quickly and specifically amplify target DNA fragments in vitro.
  • the parent strand may be the genomic DNA of a monoclonal test bacterium.
  • optical density optical density
  • the OD value mentioned in this application is the abbreviation of optical density (optical density), which represents the optical density absorbed by the test object.
  • Measure the absorbance value of the bacterial culture solution at 600nm (expressed by OD 600 ), you can measure the concentration of the bacterial culture solution to estimate the growth of bacteria, so the optical density value at 600nm can be used to express the cell density of the bacterial body, among which, The absorbance is proportional to the concentration of bacteria in the culture solution.
  • PDA medium refers to potato glucose medium, where P, D, and A are the abbreviations of Potato Dextrose Agar (Medium).
  • PDA medium is a commonly used fungus medium such as yeast, mold, and mushroom, and it is a semi-synthetic medium.
  • biocontrol bacteria or biocontrol bacteria mentioned in this application refers to one or more kinds of bacteria with biological control functions. It refers to the use of beneficial microorganisms to kill or reduce the number of pathogenic organisms to control the occurrence and development of plant diseases. Also known as "using bacteria to cure bacteria”. Biological control is an important part of the integrated pest management system. It has the advantages of not polluting the environment, non-toxic to humans and animals, and no side effects to plants. It is especially suitable for the control of soil-borne diseases.
  • Trichoderma In the ecological environment, there are many mechanisms for one kind of microorganism to control the growth of other microorganisms. Different biocontrol bacteria and the same kind of biocontrol bacteria may have different biocontrol mechanisms when interacting with different plants. Taking Trichoderma as an example, the biocontrol mechanism of biocontrol bacteria can be roughly divided into competition. For example, Trichoderma is highly adaptable to the environment, grows much faster than pathogens, can compete with pathogens for nutrition or space, and effectively utilize plant surfaces.
  • antagonism such as the non-volatile metabolites produced by Trichoderma can strongly inhibit cotton yellow
  • the growth of wilt fungus causes the hyphae of the pathogenic fungus to appear cell cytoplasmic concentration and hyphae rupture; induce resistance, such as Trichoderma viride penetrates and colonizes the cotton root epidermis and outer skin tissues, and its peroxidase activity increases ,
  • the accumulation of terpenoids can control pathogen infection more effectively than plants that are not infected by Trichoderma viride, and induce disease resistance of cotton; parasitic effect; antibiotic effect, etc.
  • Many biocontrol microorganisms play a biocontrol role through a single mechanism, and some microorganisms can work together by focusing on different mechanisms.
  • bacteria can be separated and purified from any soil where biocontrol bacteria may exist, and their biocontrol properties can be tested.
  • the tomato-rice rotation soil is taken, and the bacteria in it are separated and purified.
  • the soil diluent Weigh an appropriate amount of soil.
  • the soil mentioned here is the soil that may contain biocontrol bacteria retrieved from the wild in advance.
  • the soil can also be directly weighed in the field.
  • 10 g of soil can be weighed. Put the weighed soil into sterile water, break it, and then let it stand for more than 30 minutes to analyze the microorganisms in the soil.
  • 10 g of soil is weighed and mixed with 100 mL of sterile water. The obtained supernatant is the soil stock solution.
  • Soil stock solution was then diluted 10-fold, 102-fold, 103-fold, 104-fold, 105-fold, 106-fold and so on.
  • This step is convenient to obtain a single clone of the microorganisms in the soil.
  • test tube with 9 mL of sterile water, and mix well, so that Dilute the soil stock solution 10 times.
  • a stock solution was diluted 102-fold Soil, 103 times, 104 times, 105 times, 106 times solution. Label the test tubes as 0, 1, 2, 3, 4, 5, and 6 according to the dilution multiple of the soil stock solution.
  • the microorganisms in each test tube in step 1 are coated on the solid medium.
  • the solution is respectively sucked from the 7 test tubes made in step 1, and transferred to the surface of the bacterial solid culture medium, and the solution is evenly coated with a tool such as a spatula, a glass rod, and the like. After drying until the surface of the culture medium is relatively dry and no liquid state, the culture medium is packaged.
  • a sealing film can be used for packaging.
  • the solution of each dilution can be coated on the surface of multiple solid culture media. For example, the solution of each dilution is spread on the surface of 3 solid culture media.
  • bacteria can be cultured in a 37°C constant temperature environment, such as a 37°C constant temperature box, water bath, etc., for more than 12 hours.
  • the bacteria can also be cultured in an environment of 28°C, such as a 28°C thermostat, water bath, etc., for more than 48 hours.
  • the plate is scribed and separated.
  • the colonies obtained in the above steps were streaked and re-cultured at a suitable temperature. This operation can not only isolate the monoclonal strains, but also multiply and store the monoclonal strains, and facilitate subsequent sequencing and classification experiments.
  • the bacterial 16S universal primer can be used for preliminary identification of strain species.
  • the strain can be directly used as a template for bacterial species identification, or genomic DNA can be extracted first, and genomic DNA can be used as a template for bacterial species identification.
  • agarose gel electrophoresis is used to detect the content and quality of the extracted bacterial genomic DNA.
  • Fig. 1A is an agarose gel electrophoresis diagram of bacterial genomic DNA extracted according to an embodiment of the present application. As shown in Fig. 1A, the bands of the genomic DNA are obvious, and the concentration meets the requirements of subsequent PCR experiments.
  • PCR primers for identifying strains can be designed by themselves.
  • commonly used general primers for bacterial identification can also be selected for PCR amplification.
  • universal primers 27F and 1492R are selected for PCR amplification, wherein the DNA sequences of 27F and 1492R are as follows:
  • reaction conditions of PCR are as follows: first carry out pre-denaturation at 94°C, 5min, and then enter the cycle program, each cycle program is 94°C denaturation 30s, 55°C annealing for 30s, 72°C extension for 1min30s, for 30 cycles, and finally 72 Incubate at °C for 7min.
  • agarose gel electrophoresis is used to detect the content and quality of DNA fragments obtained after PCR using the extracted genomic DNA as a template through 16S universal primers.
  • FIG. 1B is an agarose gel electrophoresis diagram of a DNA fragment obtained by performing PCR with 16S universal primers based on the extracted bacterial genomic DNA as a template according to an embodiment of the present application. As shown in Figure 1B, the 7 groups of experiments all obtained PCR products with a single band and a concentration that met the requirements for subsequent sequencing and identification.
  • the PCR product obtained in the above experiment can be sequenced.
  • the obtained sequencing results are compared with the sequences of the existing strains, and finally the types of the identified strains are obtained.
  • input the results of sequencing to the NCBI website (https://www.ncbi.nlm.nih.gov) for blasting, and then the identified strains can be obtained.
  • Fig. 1C is a phylogenetic tree obtained by PCR amplification of the isolated strains using 16S primers according to an embodiment of the present application, and the phylogenetic tree obtained after sequence alignment, and the biocontrol bacteria identified as boron-resistant lysine Acid Bacillus (Lysinibacillus boronitolerans).
  • This strain was deposited in the China Center for Type Culture Collection on October 8, 2019, referred to as CCTCC, and the deposit number is CCTCC NO.M 2019773.
  • CCTCC boron-resistant lysine-resistant Bacillus
  • the evolutionary relationship between the boron-resistant lysine-resistant Bacillus (Lysinibacillus boronitolerans) and other bacterial species can be clearly seen.
  • the toxic medium method can be used to identify the antibacterial activity of the potential biocontrol bacteria isolated in the present application.
  • the specific operation of the toxic medium method is:
  • test pathogenic bacteria cake (diameter 8mm) in the center of the plate
  • biocontrol bacteria In this application, the antibacterial properties of biocontrol bacteria are evaluated by the following formula:
  • Mycelial growth inhibition rate (%) (control colony diameter-treated colony diameter) / (control colony diameter-0.8) ⁇ 100%
  • Figure 2 shows the growth inhibitory effect of the fermentation filtrate of Lysinibacillus boronitolerans on Magnaporthe oryzea according to an embodiment of the present application.
  • Figures 2A, 2B, and 2C are the experimental groups, in which the sterile fermentation filtrate obtained after fermentation with boron-resistant lysine Bacillus for about 24h, 60h, and 96h was added to the culture medium;
  • Figure 2D is the control group, not in The aseptic fermentation filtrate of Boron-Lysine-resistant Bacillus is added to the culture medium.
  • the dots in the middle of the medium are the target pathogens dropped in.
  • the target pathogen is Magnaporthe oryzea.
  • the ethyl acetate extraction method and the ammonium sulfate precipitation extraction method are used to crudely extract the bacterial fermentation broth with biocontrol effects, and the antibacterial activity of the crude extract is identified by the perforation method.
  • the specific operation steps of the punching method are as follows:
  • the specific operation for preparing the crude extract is as follows:
  • FIG. 3 shows a 96-hour fermentation broth crude extract of boron-lysine-resistant Bacillus fermented by an ethyl acetate extraction method prepared according to an embodiment of the present application, and its inhibitory effect on the growth of Pyricularia oryzae is tested.
  • the crude extracts of different concentrations have different degrees of inhibition on the rice blast fungus.
  • the crude extract was prepared by the ammonium sulfate precipitation method. Take 400ml of the fermentation supernatant of Boron-Lysine-resistant Bacillus under the optimal fermentation conditions for 96 hours, and add ammonium sulfate to the supernatant while stirring to make the saturation reach 50%, 60%, 70% or 80%. Precipitation overnight at 4°C. The precipitate was collected by centrifugation at 8000 rpm for 10 min. The precipitate was dissolved in PBS (pH 7.0) and the volume was adjusted to 10 ml to determine its antibacterial activity. The control solvent was PBS (pH 7.0). Fig.
  • FIG. 4 shows a 96-hour fermentation broth crude extract of boron-lysine-resistant Bacillus fermented by an ammonium sulfate precipitation method prepared according to an embodiment of the present application, and its inhibitory effect on the growth of Pyricularia oryzae is tested.
  • Figure 4 in the precipitation experiments with different ammonium sulfate saturation degrees, the crude extract of the fermentation broth with 80% ammonium sulfate saturation has the most obvious inhibitory effect on the growth of Pyricularia oryzae.
  • the sterile fermentation filtrate or crude extract of biocontrol bacteria can be combined with the medium Mix to inhibit the growth of pathogenic bacteria, thereby preventing plants from infecting pathogenic bacteria or treating plants that have been infected with pathogenic bacteria.
  • the medium is any carrier that can carry pathogenic bacteria.
  • the medium may include rice seeds, the plant itself (that is, seedlings at various stages of rice growth), and may also include the soil and water environment where the rice grows. , Even one or more of the medium for cultivating plants.
  • the pathogenic bacteria may be Magnaporthe oryzea.
  • the types of pathogenic bacteria should not be limited.
  • the bacteria and fungi that can inhibit the growth of Lysinibacillus boronitolerans in this application to a certain extent should be regarded as pathogenic bacteria mentioned in this application.
  • Lysinibacillus boronitolerans can be used as chassis microbial chassis cells for mass production of fermentation filtrate with antibacterial activity, so as to meet the demand for fungicides in agriculture, production and life.
  • the modification module further enhances the function of the fungicide for inhibiting the growth of Magnaporthe oryzea, and improves the efficiency of preparing the fungicide for inhibiting the growth of Magnaporthe oryzea.
  • Lysinibacillus boronitolerans can also be made into a fungicide to inhibit the growth of pathogenic bacteria such as Magnaporthe oryzea.
  • the bactericide includes the fermentation filtrate of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) or its crude extract and auxiliary materials.
  • the bactericide includes a fermentation broth prepared by a cell modification module based on Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) and auxiliary materials.
  • the fermentation time of the fermentation broth of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) in the fungicide is 96 hours, and the crude extract is an extract obtained by an ethyl acetate extraction method and an ammonium sulfate precipitation method.
  • the modification module is a modification module that can mass-produce one or more proteins or other substances with specific antibacterial activity.
  • the auxiliary material is one or more of water, liquid culture medium, solid culture medium, and glycerol.
  • the proportion of glycerin can be adjusted according to the storage conditions of the bactericide. For example, the volume ratio of glycerin can be 10%, 25%, 50%, 75%, etc.
  • the boron-lysine-resistant bacillus of the present application can also be mixed with other bacteria and fungi with biological control functions to prepare a preparation that has a good inhibitory effect on a variety of pathogenic bacteria.
  • the method for preventing or treating pathogenic bacteria infecting plants includes applying the aforementioned fungicide containing the fermentation broth or crude extract of Lysinibacillus boronitolerans to Mix with the medium to inhibit the growth of pathogenic bacteria on the surface of the medium.
  • the medium is any carrier that can carry pathogenic bacteria.
  • the medium may include rice seeds, the plant itself (that is, seedlings at various stages of rice growth), and may also include the soil and water environment where the rice grows.
  • the pathogenic bacteria may be Magnaporthe oryzea.
  • the types of pathogenic bacteria should not be limited.
  • the Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) of this application, as well as other biocontrol bacteria mixed with it, which can inhibit their growth to a certain extent, should be regarded as bacteria and fungi.
  • Rice blast is an important disease of rice, causing serious economic losses to agricultural production.
  • the extensive use of chemical pesticides also poses a huge threat to the environment and food.
  • Boron-resistant lysine bacillus (Lysinibacillus boronitolerans) can not only reduce the occurrence of rice blast to a large extent, but also has no threat to the ecological environment.

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Abstract

An application of Lysinibacillus boronitolerans in inhibiting growth of Magnaporthe oryzea. Lysinibacillus boronitolerans not only may greatly reduce the occurrence of and damage from rice blast, but also has no impact on the ecological environment. It is hoped that Lysinibacillus boronitolerans are will serve as high-quality chassis microorganisms in synthetic biology research, so as to bring a new direction to rice blast prevention and control.

Description

一种耐硼赖氨酸芽孢杆菌作为底盘细胞制备的稻瘟病杀菌剂Rice blast fungicide prepared by using boron-resistant lysine bacillus as chassis cells 技术领域Technical field
本发明涉及一种生防菌的合成生物学改造和应用，特别地涉及一种稻瘟病杀菌剂。The invention relates to a synthetic biology modification and application of biocontrol bacteria, in particular to a rice blast fungicide.
背景技术Background technique
“民以食为天，食以稻为先”。近5年来，我国水稻播种面积占粮食作物播种面积的27.5％左右，水稻单产比粮食作物平均单产高35.7％，稻谷产量占粮食总产的37.3％以上。水稻是我国单产最高、总产最多的粮食作物。全国65％以上人口以稻米为主食，生产的85％以上稻米作为口粮消费。一旦稻米供给不足，人民的生活就会受到影响，稻米供求的细小变化都会导致粮食价格乃至整个物价波动。水稻丰歉直接关系到粮食丰歉，水稻安全直接关系到国家粮食安全。"Food is the heaven for the people, and rice is the first for food." In the past five years, the sown area of rice in my country has accounted for about 27.5% of the sown area of food crops. The rice yield is 35.7% higher than the average yield of grain crops, and the rice output accounts for more than 37.3% of the total grain output. Rice is the food crop with the highest yield per unit and the largest total yield in my country. More than 65% of the population of the country feeds on rice as the staple food, and more than 85% of the rice produced is consumed as rations. Once the rice supply is insufficient, people's lives will be affected. Small changes in rice supply and demand will cause grain prices and even the entire price of goods to fluctuate. Rice harvest is directly related to grain harvest, and rice security is directly related to national food security.
稻瘟病是水稻重要病害之一，可引起水稻的大幅度减产，严重时可导致减产40％～50％，甚至颗粒无收。稻瘟病是真菌(如稻瘟病菌)寄生引起，病害的扩展靠分生孢子在空气中传播。病菌发育最适温度为25℃～28℃，高湿有利分生孢子形成飞散和萌发，而高湿度持续达一昼夜以上，则有利于病害发生流行。稻瘟病菌属于半知菌亚门，除感染水稻致其感染稻瘟病外，还能引起人类和一些动物皮肤病。Rice blast is one of the important diseases of rice, which can cause a substantial reduction in rice production, and in severe cases, it can lead to a reduction of 40% to 50%, or even no grain harvest. Rice blast is caused by the parasitic fungus (such as rice blast fungus), and the spread of the disease is spread by conidia in the air. The optimum temperature for the development of the pathogen is 25°C to 28°C. High humidity is good for conidia formation and germination, while high humidity for more than one day and night is good for the spread of the disease. Magnaporthe grisea belongs to the subphylum Deuteromycotina. In addition to infecting rice and causing it to infect rice blast, it can also cause skin diseases in humans and some animals.
常见的稻瘟病菌防治措施包括：Common prevention and control measures for rice blast include:
物理措施：选用排灌方便的田块；不种植感病品种；使用无病菌感染的土；定期检查病情，发现病株及时拔出等。此种防治方法需消耗大量精力，且很难完全清除土壤中的全部致病菌，一旦致病菌再次泛滥，将带来不可估量的损失。Physical measures: choose fields that are convenient for irrigation and drainage; do not plant susceptible varieties; use soil without pathogenic infection; check the condition of the disease regularly, and promptly pull out the diseased plants when found, etc. This control method requires a lot of energy, and it is difficult to completely remove all pathogenic bacteria in the soil. Once the pathogenic bacteria flood again, it will bring immeasurable losses.
药物防治：提前使用抗菌化学制剂浸种，喷洒农药等。尽管化学药剂的使用对植物病害防治带来了诸多便利以及成效，但是化学药剂的生产成本本身较高，并且使用化学药剂杀菌后，随之而来的环境污染、农产品农药残留超标以及病原菌抗药性的形成等负面效应已经引起社会的广泛关注。Drug control: use antibacterial chemicals to soak seeds in advance, spray pesticides, etc. Although the use of chemical agents has brought many conveniences and effects to the prevention and control of plant diseases, the production cost of chemical agents is inherently high, and after the use of chemical agents for sterilization, environmental pollution, excessive pesticide residues in agricultural products and pathogen resistance will follow. Negative effects such as the formation of AIDS have aroused widespread concern in the society.
因此，近年来世界各国都在努力开发可替代传统化学药剂控制植物病害的新方法。其中利用微生物及其代谢产物进行生物防治，被公认为是一种环境友好型的选择。进一步地，利用合成生物学改造具有抗稻瘟菌功能的生物***，或者对稻瘟病菌具有生长抑制功能的生防菌，将其改造成高版本模式微生物底盘细胞，使其作为最小细胞工厂生产生物农药，将成为最为快捷有效的控制植物病害途径。Therefore, in recent years, countries all over the world are striving to develop new methods that can replace traditional chemical agents to control plant diseases. Among them, the use of microorganisms and their metabolites for biological control is recognized as an environmentally friendly option. Further, using synthetic biology to transform biological systems with resistance to rice blast fungus, or biocontrol bacteria with growth inhibitory function to rice blast fungus, transform them into high-version model microbial chassis cells, making them produced as the smallest cell factory Biological pesticides will become the fastest and most effective way to control plant diseases.
发明内容Summary of the invention
针对现有技术中存在的技术问题，本发明提出了一种耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)，于2019年10月8日保藏于中国典型培养物保藏中心，简称CCTCC，保藏编号为CCTCC NO.M 2019773。In view of the technical problems in the prior art, the present invention proposes a boron-resistant lysine bacillus (Lysinibacillus boronitolerans), which was deposited in the China Center for Type Culture Collection on October 8, 2019, referred to as CCTCC, and the deposit number is CCTCC NO.M 2019773.
如上所述的耐硼赖氨酸芽孢杆菌在抑制稻瘟病菌(Magnaporthe oryzea)生长方面的应用。The application of the above-mentioned boron-resistant lysine bacillus in inhibiting the growth of Magnaporthe oryzea.
一种杀菌剂，包括：如上所述的耐硼赖氨酸芽孢杆菌发酵液(Lysinibacillus boronitolerans)以及辅料。A bactericide includes the above-mentioned boron-resistant Lysine Bacillus fermentation broth (Lysinibacillus boronitolerans) and auxiliary materials.
如上所述的杀菌剂，所述辅料为水、液体培养基、固体培养基、甘油中的一种或者多种。In the above-mentioned bactericide, the auxiliary material is one or more of water, liquid culture medium, solid culture medium, and glycerin.
如上所述的杀菌剂，其中所述的耐硼赖氨酸芽孢杆菌的发酵液为如上所述的硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)经24-96小时培养，OD 600为4.8-2.5时的发酵液。 The above bactericide, wherein the fermentation broth of Boron-resistant Lysine Bacillus is the above-mentioned Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) cultured for 24-96 hours, and the OD 600 is 4.8-2.5 hours Of fermentation broth.
如上所述的耐硼赖氨酸芽孢杆菌发酵液在制备抑制稻瘟病菌 (Magnaporthe oryzea)生长的杀菌剂的应用。The application of the above-mentioned boron-resistant lysine bacillus fermentation broth in the preparation of a fungicide for inhibiting the growth of Magnaporthe oryzea.
如上所述的耐硼赖氨酸芽孢杆菌作为底盘细胞在制备抑制稻瘟病杀菌剂中的应用。The application of the above-mentioned boron-resistant lysine bacillus as a bottom plate cell in the preparation of a fungicide for inhibiting rice blast.
一种如上所述的耐硼赖氨酸芽孢杆菌作为底盘细胞为基础的改造模块。A boron-lysine-resistant bacillus as described above is used as a chassis cell-based transformation module.
如上所述的改造模块在制备抑制稻瘟病菌(Magnaporthe oryzea)生长的杀菌剂中的应用。The application of the above-mentioned transformation module in the preparation of a fungicide for inhibiting the growth of Magnaporthe oryzea.
一种用于预防或治疗稻瘟病的方法，包括：获取如上所述的杀菌剂；以及将所述杀菌剂与介质混合。A method for preventing or treating rice blast includes: obtaining the above-mentioned fungicide; and mixing the fungicide with a medium.
如上所述的方法，其中，所述介质为携带稻瘟病菌(Magnaporthe oryzea)的载体。The method as described above, wherein the medium is a vector carrying Magnaporthe oryzea.
如上所述的方法，其中，所述载体为种子、植物、植物生长的土壤、培养植物的培养基中的一者或多者。The method as described above, wherein the carrier is one or more of a seed, a plant, the soil where the plant grows, and a medium for cultivating the plant.
如上所述的杀菌剂，其中所述耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)发酵液为耐硼赖氨酸芽孢杆菌以200ml菌液(初始OD为0.02OD 600/ml)/500ml LB培养基的装液量，在37℃下以150r/min转速培养24h、60h或96h，且菌液OD值分别达到4.8、4.0、2.5时得到的发酵产物。 The bactericide as described above, wherein the fermentation broth of Lysinibacillus boronitolerans is the fermentation broth of Lysinibacillus boronitolerans with 200ml broth (initial OD is 0.02OD 600 /ml)/500ml LB medium The fermentation product is obtained when the OD value of the bacteria liquid reaches 4.8, 4.0, 2.5, and the OD value of the bacteria liquid reaches 4.8, 4.0, 2.5, and the culture is carried out at 150r/min at 37°C for 24h, 60h or 96h.
稻瘟病是水稻的重要病害，给农业生产造成了严重的经济损失。由于大量使用化学农药给环境和食品也带来了巨大的威胁。耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)不仅可以很大程度上降低稻瘟病的发生和危害，而且对生态环境没有影响，有望作为合成生物学研究的优质底盘微生物，为稻瘟病的防治带来新的方向。Rice blast is an important disease of rice, causing serious economic losses to agricultural production. The extensive use of chemical pesticides also poses a huge threat to the environment and food. Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) can not only reduce the occurrence and damage of rice blast to a large extent, but also has no impact on the ecological environment. It is expected to be a high-quality chassis microorganism for synthetic biology research and bring to the control of rice blast. New direction.
附图说明Description of the drawings
下面，将结合附图对本发明的优选实施方式进行进一步详细的说明，其中：Hereinafter, the preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings, in which:
图1是根据本申请的一个实施例的对从土壤中分离的潜在生防菌的鉴定。 其中，图1A是从土壤中分离并纯化的细菌基因组DNA凝胶电泳分离图谱；图1B是用16S引物对基因组DNA进行特异性扩增所获得PCR产物的凝胶电泳分离图谱；图1C是通过序列比对后获得的潜在生防菌的***进化树，通过序列比对分析，鉴定所获取的潜在生防菌是耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)；Fig. 1 is the identification of potential biocontrol bacteria isolated from soil according to an embodiment of the present application. Among them, Figure 1A is a gel electrophoresis separation map of bacterial genomic DNA separated and purified from soil; Figure 1B is a gel electrophoresis separation map of PCR products obtained by specific amplification of genomic DNA with 16S primers; Figure 1C is a through The phylogenetic tree of potential biocontrol bacteria obtained after sequence alignment, through sequence comparison analysis, identified the obtained potential biocontrol bacteria as Lysinibacillus boronitolerans (Lysinibacillus boronitolerans);
图2是根据本申请的一个实施例的耐硼赖氨酸芽孢杆菌不同时期发酵液对稻瘟病菌的生长抑制作用。其中，图2A、2B、2C是实验组，在其培养基中分别加入了耐硼赖氨酸芽孢杆菌发酵24h、60h、96h后获得的无菌发酵滤液；图2D是对照组，未在其培养基中加入耐硼赖氨酸芽孢杆菌的无菌发酵滤液；Fig. 2 shows the growth inhibitory effect of the fermentation broth of boron-lysine-resistant Bacillus at different stages on the rice blast fungus according to an embodiment of the present application. Among them, Figures 2A, 2B, and 2C are the experimental groups, in which the sterile fermentation filtrate obtained after fermentation with boron-resistant lysine Bacillus for 24h, 60h, and 96h was added to the culture medium; Figure 2D is the control group. Add sterile fermentation filtrate of Boron-Lysine-resistant Bacillus to the medium;
图3为根据本申请的一个实施例的耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)96h发酵液的不同浓度乙酸乙酯提取物对稻瘟病菌(Magnaporthe oryzea)的抑菌活性检测。其中，粗提物的浓度分别为1mg/ml、5mg/ml、10mg/ml和15mg/ml；以及Fig. 3 shows the antibacterial activity detection of different concentrations of ethyl acetate extracts of 96h fermentation broth of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) against Magnaporthe oryzea according to an embodiment of the present application. Wherein, the concentration of the crude extract is 1mg/ml, 5mg/ml, 10mg/ml and 15mg/ml; and
图4为根据本申请的一个实施例的耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)96h发酵液以不同饱和度的硫酸铵沉淀所获得提取物对稻瘟病菌(Magnaporthe oryzea)的抑菌活性检测。Figure 4 shows the detection of antibacterial activity of extracts obtained by precipitation of ammonium sulfate with different saturations in 96h fermentation broth of Lysinibacillus boronitolerans according to an embodiment of the present application against Magnaporthe oryzea .
具体实施方式Detailed ways
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments These are a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.
在以下的详细描述中，可以参看作为本申请一部分用来说明本申请的特定实施例的各个说明书附图。本申请的各个特定实施例在以下进行了足够详细的 描述，使得具备本领域相关知识和技术的普通技术人员能够实施本申请的技术方案。应当理解，还可以利用其它实施例或者对本申请的实施例进行结构、逻辑的改变。In the following detailed description, reference may be made to the various drawings of the specification that are part of this application to illustrate specific embodiments of the application. The specific embodiments of the present application are described in sufficient detail below to enable those of ordinary skill with relevant knowledge and technology in the field to implement the technical solutions of the present application. It should be understood that other embodiments may also be used or structural and logical changes to the embodiments of the present application may be made.
本领域技术人员应当理解，当陈述细菌经发酵、培养至一定浓度时，所说的浓度为一定数值范围内的浓度，例如当以OD值表示浓度时，申请中若出现OD 600为4.8，则实际OD值约为4.8，如OD 600值为4.8±0.6。将细菌发酵、培养至一定程度所需时间均为不能精确至分、秒的确定的时间，且所需时间与细菌发酵、培养的环境温度、培养基种类等均有关系，所以本申请中出现的与细菌发酵、培养、生长等相关的时间，均为大约的时间，而不是确定的时间。 Those skilled in the art should understand that when it is stated that bacteria are fermented and cultured to a certain concentration, the said concentration is a concentration within a certain numerical range. For example, when the concentration is expressed by the OD value, if an OD 600 of 4.8 appears in the application, then The actual OD value is about 4.8, such as the OD 600 value is 4.8 ± 0.6. The time required to ferment and cultivate the bacteria to a certain level is a certain time that cannot be accurate to minutes and seconds, and the required time is related to the environmental temperature of bacterial fermentation, culture, and the type of culture medium, so it appears in this application The time related to bacterial fermentation, cultivation, growth, etc., are approximate times, not definite times.
本申请中出现的一些名词具有如下释义：Some nouns appearing in this application have the following interpretations:
本申请所说的发酵液是指菌经发酵后产生的含有发酵产物的液体，例如耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)发酵液为耐硼赖氨酸芽孢杆菌以200ml菌液(初始OD约为0.02OD 600/ml)/500ml LB培养基的装液量，在37℃下以150r/min转速培养约24h、60h或96h得到的发酵产物。耐硼赖氨酸芽孢杆菌发酵时间约为24、60h和96h时，菌液OD值分别约为4.8、4.0和2.5(OD值随着发酵时间的延长而降低)。发酵结束后，离心去除沉淀，收集发酵上清液，然后用0.22μm的滤膜过滤，即得到无菌的发酵滤液。 The fermentation broth mentioned in this application refers to the fermentation product-containing liquid produced by bacteria after fermentation. For example, the fermentation broth of Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) is a 200ml broth (initial OD). About 0.02OD 600 /ml)/500ml LB medium filling volume, the fermentation product obtained by culturing at 37°C at 150r/min for about 24h, 60h or 96h. When the fermentation time of boron-resistant lysine bacillus was about 24, 60h and 96h, the OD value of the bacterial solution was about 4.8, 4.0 and 2.5 (OD value decreased with the extension of fermentation time). After the fermentation, the precipitate is removed by centrifugation, the fermentation supernatant is collected, and then filtered with a 0.22 μm filter membrane to obtain a sterile fermentation filtrate.
本申请所说的含毒介质法又叫生长速率法,是杀菌剂毒力测定的常规方法之一,适用于不长孢子而菌丝生长较快的真菌。通过菌落生长的速度快慢可以衡量药剂的毒力大小。含毒介质法是将供试药剂与培养基混合,以培养基上菌落的生长速度来衡量药剂的毒力大小。一般多用于那些不产孢子或孢子量少而菌丝较密的真菌。菌落生长速率一般通过菌落达到一个给定的大小所需要的时间(天或小时)，或者在单位时间内菌落直径的大小表示。The toxic medium method mentioned in this application is also called the growth rate method, which is one of the conventional methods for the determination of the virulence of fungicides, and is suitable for fungi that do not grow spores but have faster hyphae growth. The virulence of the agent can be measured by the growth rate of the colony. Toxic medium method is to mix the test agent with the culture medium, and measure the virulence of the medicine by the growth rate of the colony on the culture medium. Generally used for fungi that do not produce spores or have a small amount of spores and dense hyphae. The colony growth rate is generally expressed by the time (days or hours) required for the colony to reach a given size, or the size of the colony diameter per unit time.
本申请所说的打孔法，是指用已灭菌的打孔器或钢管等在试验平板上打孔，然后往孔中注入一定量的待检测样品，培养一段时间后测定其抑菌圈大小 的一种方法。The perforating method mentioned in this application refers to perforating a test plate with a sterilized perforator or steel pipe, and then injecting a certain amount of the sample to be tested into the hole, and measuring the inhibition zone after incubating for a period of time. A way of size.
本文所说的萃取法是指利用化合物在两种互不相溶(或微溶)的溶剂中溶解度或分配系数的不同，使化合物从一种溶剂内转移到另外一种溶剂中。经过反复多次萃取，将绝大部分的化合物提取出来的方法。乙酸乙酯属于中极性溶剂，可以从细菌的发酵滤液中萃取多种物质，例如具有较小极性的分子(如葡萄糖或带糖的苷类)、特小极性的分子(如某些石蜡)、含有盐结构的分子(如氨基酸)等。The extraction method mentioned herein refers to the use of the difference in solubility or partition coefficient of a compound in two immiscible (or slightly soluble) solvents to transfer the compound from one solvent to another. After repeated extractions, most of the compounds are extracted. Ethyl acetate is a medium polar solvent, which can extract a variety of substances from the fermentation filtrate of bacteria, such as molecules with less polarity (such as glucose or glycosides), molecules with very small polarity (such as certain Paraffin), molecules containing salt structures (such as amino acids), etc.
本申请所说的硫酸铵沉淀是指用不同浓度硫酸铵溶液对蛋白质进行沉淀和分离的技术。常用于分离免疫球蛋白。硫酸铵沉淀法可用于从大量粗制剂中浓缩和部分纯化蛋白质。高浓度的盐离子在蛋白质溶液中可与蛋白质竞争水分子，从而破坏蛋白质表面的水化膜，降低其溶解度，使之从溶液中沉淀出来。各种蛋白质的溶解度不同，因而可利用不同浓度的盐溶液来沉淀不同的蛋白质。这种方法称之为盐析。盐浓度通常用饱和度来表示。硫酸铵因其溶解度大，温度系数小和不易使蛋白质变性而应用最广。The ammonium sulfate precipitation mentioned in this application refers to the technique of using different concentrations of ammonium sulfate solutions to precipitate and separate proteins. Commonly used to separate immunoglobulins. The ammonium sulfate precipitation method can be used to concentrate and partially purify proteins from a large number of crude preparations. The high concentration of salt ions in the protein solution can compete with the protein for water molecules, thereby destroying the hydration film on the surface of the protein, reducing its solubility, and making it precipitate out of the solution. The solubility of various proteins is different, so different concentrations of salt solutions can be used to precipitate different proteins. This method is called salting out. Salt concentration is usually expressed in terms of saturation. Ammonium sulfate is the most widely used because of its high solubility, low temperature coefficient and difficulty in denaturing protein.
本申请所说的16S引物鉴定细菌是指运用PCR及序列比对等方法，确定待测细菌的种类。16S rDNA是细菌染色体上编码16S rRNA相对应的DNA序列，存在于所有细菌染色体基因中，它的内部结构由保守区及可变区两部分组成。其分子内存在的可变区，显示出细菌不同分类等级水平上的特异性。The identification of bacteria with 16S primers in this application refers to the use of methods such as PCR and sequence comparison to determine the type of bacteria to be tested. 16S rDNA is a DNA sequence corresponding to 16S rRNA on bacterial chromosomes, which is present in all bacterial chromosomal genes. Its internal structure is composed of conserved regions and variable regions. The variable regions within the molecule show the specificity of bacteria at the level of different classification levels.
细菌中包括三种核糖体RNA，分别为5S、16S和23S rRNA。其中，5S rRNA虽易分析，但核苷酸数量太少，仅几十个核苷酸组成，导致遗传信息不足而不能用于分类研究；23S rRNA则因其分子量太大，所含有的核苷酸数量几乎是16S rRNA的两倍，分析较困难而不选择其进行分类研究。通常将16S rRNA用于细菌分类研究。There are three types of ribosomal RNA in bacteria, namely 5S, 16S and 23S rRNA. Among them, 5S rRNA is easy to analyze, but the number of nucleotides is too small, consisting of only a few dozen nucleotides, resulting in insufficient genetic information and cannot be used for classification research; 23S rRNA is too large for its molecular weight and contains nucleosides. The amount of acid is almost twice that of 16S rRNA, and it is difficult to analyze and not select it for classification research. 16S rRNA is usually used for bacterial classification research.
首先，16S rRNA普遍存在于原核生物(真核生物中其同源分子是18S rRNA)中。rRNA参与生物蛋白质的合成过程，其功能是任何生物都必不 可少的，而且在生物进化的漫长历程中保持不变，可看作为生物演变的时间钟。其次，在16S rRNA分子中，既含有高度保守的序列区域，又有中度保守和高度变化的序列区域，因而它适用于进化距离不同的各类生物亲缘关系的研究。第三，16S rRNA的相对分子量大小适中，约2kb个核苷酸，便于序列分析。因此，它可以作为测量各类生物进化和亲缘关系的良好工具。First, 16S rRNA is ubiquitous in prokaryotes (the homologous molecule in eukaryotes is 18S rRNA). rRNA participates in the process of biological protein synthesis, its function is indispensable to any organism, and it remains unchanged during the long course of biological evolution, which can be regarded as the time clock of biological evolution. Secondly, 16S rRNA molecules contain both highly conserved sequence regions and moderately conserved and highly variable sequence regions. Therefore, it is suitable for the study of various biological relationships with different evolutionary distances. Third, the relative molecular weight of 16S rRNA is moderate, about 2kb nucleotides, which is convenient for sequence analysis. Therefore, it can be used as a good tool to measure the evolution and genetic relationship of various organisms.
16S rRNA的编码基因是16S rDNA，直接从细菌中提取16S rRNA很困难，且提取的RNA易降解，不易保存等，因而通常利用16S rDNA鉴定细菌的种类。The coding gene of 16S rRNA is 16S rDNA. It is difficult to directly extract 16S rRNA from bacteria, and the extracted RNA is easily degraded and difficult to store. Therefore, 16S rDNA is usually used to identify the type of bacteria.
本申请所说PCR即聚合酶链式反应，是指在DNA聚合酶催化下，以母链DNA为模板，以特定引物为延伸起点，通过变性、退火、延伸等步骤，体外复制出与母链模板DNA互补的子链DNA的过程。PCR是一项体外合成放大技术，能快速特异地在体外扩增目的DNA片段。在本申请的实施例中，母链可以为单克隆待测细菌的基因组DNA。The PCR, or polymerase chain reaction mentioned in this application, means that under the catalysis of DNA polymerase, the parent strand DNA is used as a template, and a specific primer is used as the starting point for extension. Through the steps of denaturation, annealing, extension, etc., it replicates with the parent strand in vitro. The process of DNA complementary to the template DNA. PCR is an in vitro synthetic amplification technology that can quickly and specifically amplify target DNA fragments in vitro. In the embodiments of the present application, the parent strand may be the genomic DNA of a monoclonal test bacterium.
本申请所述的OD值是optical density(光密度)的缩写，表示被检测物吸收的光密度。测量细菌培养液在600nm处的吸光值(用OD 600表示)，可以测量细菌培养液的浓度，从而估计细菌的生长情况，所以600nm处的光密度值可以用于表示菌体细胞密度，其中，吸光值正比于培养液中的细菌的浓度。 The OD value mentioned in this application is the abbreviation of optical density (optical density), which represents the optical density absorbed by the test object. Measure the absorbance value of the bacterial culture solution at 600nm (expressed by OD 600 ), you can measure the concentration of the bacterial culture solution to estimate the growth of bacteria, so the optical density value at 600nm can be used to express the cell density of the bacterial body, among which, The absorbance is proportional to the concentration of bacteria in the culture solution.
本申请所述PDA培养基，是指马铃薯葡萄糖培养基，其中P、D、A即为Potato Dextrose Agar(Medium)的缩写。PDA培养基是一种常用的酵母菌、霉菌、蘑菇等真菌培养基，是一种半合成培养基。The PDA medium described in this application refers to potato glucose medium, where P, D, and A are the abbreviations of Potato Dextrose Agar (Medium). PDA medium is a commonly used fungus medium such as yeast, mold, and mushroom, and it is a semi-synthetic medium.
本申请所说的生防细菌或者生防菌，是指具有生物防治功能的一种或者多种细菌。是指利用有益微生物杀灭或压低病原生物数量以控制植物病害发生、发展的一类措施。又称“以菌治菌”。生物防治是病虫害综合治理体系的重要一环。它具有不污染环境、对人畜无毒、对植物无副作用等优点,对土传病害的控制尤其适用。The biocontrol bacteria or biocontrol bacteria mentioned in this application refers to one or more kinds of bacteria with biological control functions. It refers to the use of beneficial microorganisms to kill or reduce the number of pathogenic organisms to control the occurrence and development of plant diseases. Also known as "using bacteria to cure bacteria". Biological control is an important part of the integrated pest management system. It has the advantages of not polluting the environment, non-toxic to humans and animals, and no side effects to plants. It is especially suitable for the control of soil-borne diseases.
在生态环境中，一种微生物控制其他微生物生长的作用机制有很多种，不同的生防菌，以及同种生防菌在与不同植物作用时，可能有不同的生防机制。以木霉菌为例，生防菌的生防机制大体可分为竞争作用，如木霉菌对环境的适应性强，生长速度远比病原菌快，能与病原菌竞争营养或空间，有效地利用植物表面或侵入点附近的浓度营养物质，迅速占领空间吸收营养，占领病原菌的入侵位点而不为病原菌的入侵留下空隙；拮抗作用，如木霉菌可以产生的非挥发性代谢物能够强烈抑制棉花黄萎病菌生长，使病原菌菌丝出现细胞原生质浓缩和菌丝断裂等现象；诱导抗性作用，如绿色木霉穿透并定植在棉花根表皮和外皮层组织中，其过氧化物酶活性升高，萜类化合物积累，比没有被绿色木霉侵染的植株更有效地控制了病原菌感染，诱导了棉花的抗病性；寄生作用；抗生作用等。许多生防微生物是通过某一种单一机制起到生防作用的，也有部分微生物可通过集中不同机制而联合发挥功能。In the ecological environment, there are many mechanisms for one kind of microorganism to control the growth of other microorganisms. Different biocontrol bacteria and the same kind of biocontrol bacteria may have different biocontrol mechanisms when interacting with different plants. Taking Trichoderma as an example, the biocontrol mechanism of biocontrol bacteria can be roughly divided into competition. For example, Trichoderma is highly adaptable to the environment, grows much faster than pathogens, can compete with pathogens for nutrition or space, and effectively utilize plant surfaces. Or the concentration of nutrients near the entry point, quickly occupy the space to absorb nutrients, occupy the invasion site of pathogens without leaving a gap for the invasion of pathogens; antagonism, such as the non-volatile metabolites produced by Trichoderma can strongly inhibit cotton yellow The growth of wilt fungus causes the hyphae of the pathogenic fungus to appear cell cytoplasmic concentration and hyphae rupture; induce resistance, such as Trichoderma viride penetrates and colonizes the cotton root epidermis and outer skin tissues, and its peroxidase activity increases , The accumulation of terpenoids can control pathogen infection more effectively than plants that are not infected by Trichoderma viride, and induce disease resistance of cotton; parasitic effect; antibiotic effect, etc. Many biocontrol microorganisms play a biocontrol role through a single mechanism, and some microorganisms can work together by focusing on different mechanisms.
本申请通过从土壤中分离、纯化出约100多株可能具有生防活性的细菌，以本实验室中现存的主要真菌病原菌作为靶标，检测分离细菌的生防特性。In this application, more than 100 strains of bacteria that may have biocontrol activity are isolated and purified from the soil, and the main fungal pathogens existing in the laboratory are used as targets to detect the biocontrol properties of the isolated bacteria.
实施例1土壤菌株的分离、纯化Example 1 Isolation and purification of soil strains
根据本申请的一个实施例，可以从可能存在生防细菌的任何土壤中分离、纯化细菌，检测其生防特性。根据本申请的一个实施例，取番茄-水稻轮作土壤，并对其中的细菌进行分离、纯化。According to an embodiment of the present application, bacteria can be separated and purified from any soil where biocontrol bacteria may exist, and their biocontrol properties can be tested. According to an embodiment of the present application, the tomato-rice rotation soil is taken, and the bacteria in it are separated and purified.
1.制备土壤稀释液。称取适量的土壤。此处所说土壤为提前从野外取回的可能含有生防菌的土壤。根据本申请的一个实施例，也可以直接在野外称取土壤。根据本申请的一个实施例，可以称取10g土壤。将称取的土壤放入无菌水中，打碎，后静置30分钟以上，充分析出土壤中的微生物。根据本申请的一个实施例，称取10g土壤，将其混于100mL无菌水中。得到的上清液即为土 壤原液。1. Prepare soil diluent. Weigh an appropriate amount of soil. The soil mentioned here is the soil that may contain biocontrol bacteria retrieved from the wild in advance. According to an embodiment of the present application, the soil can also be directly weighed in the field. According to an embodiment of the present application, 10 g of soil can be weighed. Put the weighed soil into sterile water, break it, and then let it stand for more than 30 minutes to analyze the microorganisms in the soil. According to an embodiment of the present application, 10 g of soil is weighed and mixed with 100 mL of sterile water. The obtained supernatant is the soil stock solution.
然后将土壤原液稀释10倍、10 2倍、10 3倍、10 4倍、10 5倍、10 6倍等。根据本申请的一个实施例，按上述方式稀释，即可得到1g、10 -1g、10 -2g、10 -3g、10 -4g、10 -5g、10 -6g等土壤中微生物含量。此步骤便于获得土壤中微生物的单克隆。例如，根据本申请的一个实施例，吸取10mL土壤原液于0号试管中，再从本试管中吸取1mL土壤原液，混合于装有9mL无菌水的1号试管中，充分混合，是以将土壤原液稀释10倍。依次操作，即可得到土壤原液稀释10 2倍、10 3倍、10 4倍、10 5倍、10 6倍的溶液。按土壤原液的稀释倍数，将试管分别标号为0，1，2，3，4，5，6。 Soil stock solution was then diluted 10-fold, 102-fold, 103-fold, 104-fold, 105-fold, 106-fold and so on. According to an embodiment of the present application, dilute in the above-mentioned manner to obtain 1 g, 10 -1 g, 10 -2 g, 10 -3 g, 10 -4 g, 10 -5 g, 10 -6 g and other soils Microbial content. This step is convenient to obtain a single clone of the microorganisms in the soil. For example, according to an embodiment of the present application, suck 10 mL of the soil stock solution in a No. 0 test tube, and then suck 1 mL of the soil stock solution from this test tube, mix it in a No. 1 test tube with 9 mL of sterile water, and mix well, so that Dilute the soil stock solution 10 times. Operating sequentially, to obtain a stock solution was diluted 102-fold Soil, 103 times, 104 times, 105 times, 106 times solution. Label the test tubes as 0, 1, 2, 3, 4, 5, and 6 according to the dilution multiple of the soil stock solution.
2.涂板。即，将步骤1中各试管中的微生物，涂布于固体培养基上。根据本申请的一个实施例，从步骤1制作的7个试管中分别吸取溶液，移送至细菌固体培养基表面，并用刮刀、玻璃棒等工具将溶液涂布均匀。晾晒至培养基表面较干燥，无液体状态后，将培养基封装。根据本申请的一个实施例，可利用封口膜封装。根据本申请的一个实施例，根据实验及统计的需求，可以将每个稀释度的溶液涂布于多个固体培养基表面。例如将每个稀释度的溶液均涂布于3个固体培养基表面。2. Paint the board. That is, the microorganisms in each test tube in step 1 are coated on the solid medium. According to an embodiment of the present application, the solution is respectively sucked from the 7 test tubes made in step 1, and transferred to the surface of the bacterial solid culture medium, and the solution is evenly coated with a tool such as a spatula, a glass rod, and the like. After drying until the surface of the culture medium is relatively dry and no liquid state, the culture medium is packaged. According to an embodiment of the present application, a sealing film can be used for packaging. According to an embodiment of the present application, according to experimental and statistical requirements, the solution of each dilution can be coated on the surface of multiple solid culture media. For example, the solution of each dilution is spread on the surface of 3 solid culture media.
3.微生物培养。将涂布细菌的固体培养基放置于合适环境中，待长出可见单克隆菌落后，取出。根据本申请的一个实施例，可以将细菌放置37℃恒温环境中培养，如37℃恒温箱，水浴等，培养12小时以上。也可以将细菌放置在28℃环境中培养，如28℃恒温箱，水浴等，培养48小时以上。3. Microbial culture. Place the bacteria-coated solid culture medium in a suitable environment, and take it out after the visible monoclonal bacteria grow. According to an embodiment of the present application, bacteria can be cultured in a 37°C constant temperature environment, such as a 37°C constant temperature box, water bath, etc., for more than 12 hours. The bacteria can also be cultured in an environment of 28°C, such as a 28°C thermostat, water bath, etc., for more than 48 hours.
4.计数及菌落描述。计算每个固体培养基表面的菌落个数，观察其菌落形态特征，并记录结果。例如，0号试管中为直接吸取的10mL土壤原液，将其 涂覆于固体培养基上，培养得到的菌落数目即为1g土壤中细菌数；1号试管中液体为将0号试管中溶液稀释10倍得到的，所以将其涂覆于固体培养基上，培养得到的菌落数目即为0.1g土壤中细菌数，其他试管以此类推。4. Count and description of colonies. Count the number of colonies on the surface of each solid medium, observe the morphological characteristics of the colonies, and record the results. For example, in test tube 0, 10 mL of soil stock solution is directly sucked and coated on a solid medium, and the number of colonies obtained by culturing is the number of bacteria in 1 g of soil; the liquid in test tube 1 is the dilution of the solution in test tube 0 It is obtained by 10 times, so it is coated on a solid medium, and the number of colonies obtained by culturing is the number of bacteria in 0.1g of soil, and other test tubes can be deduced by analogy.
5.平板划线分离。将上述步骤中得到的菌落划线分离，置于适合温度重新培养。如此操作，不仅可以将单克隆菌种分离，还可以对单克隆菌种扩繁和保存，且利于后续测序、分类试验。5. The plate is scribed and separated. The colonies obtained in the above steps were streaked and re-cultured at a suitable temperature. This operation can not only isolate the monoclonal strains, but also multiply and store the monoclonal strains, and facilitate subsequent sequencing and classification experiments.
6.菌种保存和鉴定。6. Bacteria preservation and identification.
实施例2鉴定菌株种类Example 2 Identification of strains
根据本申请的一个实施例，可以用细菌16S通用引物对菌株种类进行初步鉴定。根据本申请的一个实施例，可以直接将菌株作为模板进行菌种鉴定，也可以先提取基因组DNA，将基因组DNA作为模板进行菌种鉴定。根据本申请的一个实施例，使用琼脂糖凝胶电泳检测所提取的细菌基因组DNA含量、品质。图1A为根据本申请的一个实施例所提取的细菌基因组DNA琼脂糖凝胶电泳图，如图1A所示，基因组DNA的条带明显，浓度符合后续PCR实验的需求。According to an embodiment of the present application, the bacterial 16S universal primer can be used for preliminary identification of strain species. According to an embodiment of the present application, the strain can be directly used as a template for bacterial species identification, or genomic DNA can be extracted first, and genomic DNA can be used as a template for bacterial species identification. According to an embodiment of the present application, agarose gel electrophoresis is used to detect the content and quality of the extracted bacterial genomic DNA. Fig. 1A is an agarose gel electrophoresis diagram of bacterial genomic DNA extracted according to an embodiment of the present application. As shown in Fig. 1A, the bands of the genomic DNA are obvious, and the concentration meets the requirements of subsequent PCR experiments.
1.设计选择引物进行16S rDNA的PCR扩增。根据本申请的一个实施例，可自行设计鉴定菌株种类的PCR引物。根据本申请的一个实施例，也可以选择常用的细菌鉴定通用引物进行PCR扩增。根据本申请的一个实施例，选用通用引物27F和1492R进行PCR扩增，其中27F和1492R的DNA序列如下：1. Design and select primers for PCR amplification of 16S rDNA. According to an embodiment of the present application, PCR primers for identifying strains can be designed by themselves. According to an embodiment of the present application, commonly used general primers for bacterial identification can also be selected for PCR amplification. According to an embodiment of the present application, universal primers 27F and 1492R are selected for PCR amplification, wherein the DNA sequences of 27F and 1492R are as follows:
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；27F: 5’-AGAGTTTGATCCTGGCTCAG-3’;
1492R：5’-GGTTACCTTGTTACGACTT-3’。1492R: 5'-GGTTACCTTGTTACGACTT-3'.
根据选取的不同DNA聚合酶，选择适合的PCR反应体系以及反应条件。 本申请不限定任何种类DNA聚合酶的使用，也不限定使用同一种DNA聚合酶后PCR反应体系与反应条件。例如，选用Taq DNA聚合酶进行PCR扩增时，可参考如表1所列的反应体系：According to the different selected DNA polymerases, select the appropriate PCR reaction system and reaction conditions. This application does not limit the use of any kind of DNA polymerase, nor does it limit the PCR reaction system and reaction conditions after using the same DNA polymerase. For example, when using Taq DNA polymerase for PCR amplification, you can refer to the reaction system listed in Table 1:
表1.PCR体系表(12μL)Table 1. PCR system table (12μL)
Figure PCTCN2020097735-appb-000001
Figure PCTCN2020097735-appb-000001
PCR的反应条件为：首先进行预变性，条件为94℃、5min，然后进入循环程序，每个循环程序是94℃变性30s，55℃退火30s，72℃延伸1min30s，进行30个循环，最后72℃保温7min。The reaction conditions of PCR are as follows: first carry out pre-denaturation at 94℃, 5min, and then enter the cycle program, each cycle program is 94℃ denaturation 30s, 55℃ annealing for 30s, 72℃ extension for 1min30s, for 30 cycles, and finally 72 Incubate at ℃ for 7min.
根据本申请的一个实施例，使用琼脂糖凝胶电泳检测以提取的基因组DNA为模板经16S通用引物进行PCR后得到的DNA片段的含量与品质。图1B为根据本申请的一个实施例为根据所提取的细菌基因组DNA为模板经16S通用引物进行PCR后得到的DNA片段的琼脂糖凝胶电泳图。如图1B所示，7组实验均得到条带单一、浓度符合后续测序鉴定需求的PCR产物。According to an embodiment of the present application, agarose gel electrophoresis is used to detect the content and quality of DNA fragments obtained after PCR using the extracted genomic DNA as a template through 16S universal primers. FIG. 1B is an agarose gel electrophoresis diagram of a DNA fragment obtained by performing PCR with 16S universal primers based on the extracted bacterial genomic DNA as a template according to an embodiment of the present application. As shown in Figure 1B, the 7 groups of experiments all obtained PCR products with a single band and a concentration that met the requirements for subsequent sequencing and identification.
2.菌株种类鉴定。根据本申请的一个实施例，可以对上述实验得到的PCR产物进行测序处理。将得到的测序结果与现有菌种序列进行比对，最终得到所鉴定菌株的种类。例如，将测序得到的结果输入至NCBI网站(https://www.ncbi.nlm.nih.gov)进行blast，即可得到所鉴定菌株种类。2. Identification of strains. According to an embodiment of the present application, the PCR product obtained in the above experiment can be sequenced. The obtained sequencing results are compared with the sequences of the existing strains, and finally the types of the identified strains are obtained. For example, input the results of sequencing to the NCBI website (https://www.ncbi.nlm.nih.gov) for blasting, and then the identified strains can be obtained.
图1C是根据本申请的一个实施例，采用16S引物对所分离的菌株进行PCR扩增获得16S rDNA，并通过序列比对后获得的***进化树，鉴定得到的生防 菌是耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)。该菌种已于2019年10月8日保藏于中国典型培养物保藏中心，简称CCTCC，保藏编号为CCTCC NO.M 2019773。根据图1C中所示的***进化树，可以很清晰地看出本申请的耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)与其他细菌种属的进化关系。Fig. 1C is a phylogenetic tree obtained by PCR amplification of the isolated strains using 16S primers according to an embodiment of the present application, and the phylogenetic tree obtained after sequence alignment, and the biocontrol bacteria identified as boron-resistant lysine Acid Bacillus (Lysinibacillus boronitolerans). This strain was deposited in the China Center for Type Culture Collection on October 8, 2019, referred to as CCTCC, and the deposit number is CCTCC NO.M 2019773. According to the phylogenetic tree shown in Fig. 1C, the evolutionary relationship between the boron-resistant lysine-resistant Bacillus (Lysinibacillus boronitolerans) and other bacterial species can be clearly seen.
实施例3分离细菌的生防特性鉴定Example 3 Identification of biocontrol characteristics of isolated bacteria
根据本申请的一个实施例，可采用含毒介质法对本申请分离出的潜在生防菌的抑菌活性进行鉴定。根据本申请的一个实施例，含毒介质法的具体操作为：According to an embodiment of the present application, the toxic medium method can be used to identify the antibacterial activity of the potential biocontrol bacteria isolated in the present application. According to an embodiment of the present application, the specific operation of the toxic medium method is:
1.配置PDA固体培养基，并将其冷却至55℃左右；或者将已凝固的PDA培养基加热融化并冷却至55℃左右；1. Configure the PDA solid medium and cool it to about 55°C; or heat and melt the solidified PDA medium and cool it to about 55°C;
2.挑取耐硼赖氨酸芽孢杆菌单菌落接种于LB液体培养基中，37℃，150r/min过夜培养，次日转接菌液，以200ml菌液/500ml LB培养基的装液量，至终浓度为0.02OD 600/ml，150r/min转速，置于恒温摇床37℃培养约24h、60h和96h，使得OD值分别达到4.8、4.0和2.5左右。在不同时间点分别收集发酵产物，置于离心机中，以8000r/min离心去除沉淀，获得发酵上清液。然后用0.22μm的滤膜过滤发酵上清液，即得到无菌的发酵滤液； 2. Pick a single colony of boron-resistant lysine bacillus and inoculate it in LB liquid medium, cultivate overnight at 37°C, 150r/min, transfer the bacterial solution the next day, and use 200ml bacterial solution/500ml LB medium to fill the volume. , To the final concentration of 0.02OD 600 /ml, 150r/min rotation speed, placed in a constant temperature shaker at 37 ℃ for about 24h, 60h and 96h, so that the OD value reached 4.8, 4.0 and 2.5, respectively. The fermentation products were collected at different time points, placed in a centrifuge, and centrifuged at 8000r/min to remove the precipitate to obtain the fermentation supernatant. Then filter the fermentation supernatant with a 0.22μm filter membrane to obtain a sterile fermentation filtrate;
3.将发酵滤液与PDA培养基以1：9的体积比混合，即发酵液占10％，制成含拮抗菌发酵液的含药平板；3. Mix the fermentation filtrate and the PDA medium in a volume ratio of 1:9, that is, the fermentation broth accounts for 10%, to make a drug-containing plate containing the antagonistic antibacterial fermentation broth;
4.待混有发酵液的培养基冷却后，在平板中央接种1个供试病原菌菌饼(直径为8mm)；4. After the culture medium mixed with the fermentation broth is cooled, inoculate 1 test pathogenic bacteria cake (diameter 8mm) in the center of the plate;
5.将接种后的培养基置于28℃恒温培养箱进行培养。5. Place the inoculated medium in a 28°C constant temperature incubator for cultivation.
以不含发酵液的PDA培养基平板作为对照，以上步骤1-4重复至少三次， 即做至少三个生防菌对特定细菌抑菌性测试实验。Take the PDA medium plate without fermentation broth as a control, repeat the above steps 1-4 at least three times, that is, do at least three antibacterial test experiments of biocontrol bacteria against specific bacteria.
本申请中，通过以下公式评价生防菌的抑菌特性：In this application, the antibacterial properties of biocontrol bacteria are evaluated by the following formula:
菌丝生长抑制率(％)＝(对照菌落直径-处理菌落直径)/(对照菌落直径-0.8)×100％Mycelial growth inhibition rate (%) = (control colony diameter-treated colony diameter) / (control colony diameter-0.8) × 100%
图2所示为根据本申请的一个实施例的耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)发酵滤液对稻瘟病菌(Magnaporthe oryzea)的生长抑制作用。其中，图2A、2B、2C是实验组，在其培养基中分别加入了耐硼赖氨酸芽孢杆菌发酵约24h、60h、96h后获得的无菌发酵滤液；图2D是对照组，未在其培养基中加入耐硼赖氨酸芽孢杆菌的无菌发酵滤液。培养基中间的圆点为滴入的靶标病原菌。根据本申请的一个实施例，靶标病原菌为稻瘟病菌(Magnaporthe oryzea)。Figure 2 shows the growth inhibitory effect of the fermentation filtrate of Lysinibacillus boronitolerans on Magnaporthe oryzea according to an embodiment of the present application. Among them, Figures 2A, 2B, and 2C are the experimental groups, in which the sterile fermentation filtrate obtained after fermentation with boron-resistant lysine Bacillus for about 24h, 60h, and 96h was added to the culture medium; Figure 2D is the control group, not in The aseptic fermentation filtrate of Boron-Lysine-resistant Bacillus is added to the culture medium. The dots in the middle of the medium are the target pathogens dropped in. According to an embodiment of the present application, the target pathogen is Magnaporthe oryzea.
结合图2及上述公式可以明显看出，稻瘟病菌的生长明显被耐硼赖氨酸芽孢杆菌发酵滤液抑制。经测算，耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)发酵约24h、60h和96h的发酵液对稻瘟病菌(Magnaporthe oryzea)的拮抗效率分别约为27.14％、42.85％、57.69％。根据本申请的多次实验验证，耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)的发酵时间在96h，OD为2.5左右时，其发酵滤液可有效抑制稻瘟病菌(Magnaporthe oryzea)生长，拮抗效率达到约57.69％。Combining Figure 2 and the above formula, it can be clearly seen that the growth of Pyricularia oryzae is obviously inhibited by the fermentation filtrate of Boron-Lysine-resistant Bacillus. According to calculations, the antagonistic efficiency of the fermentation broth of Lysinibacillus boronitolerans for about 24h, 60h and 96h against Magnaporthe oryzea is about 27.14%, 42.85% and 57.69%, respectively. According to multiple experiments in this application, the fermentation time of Lysinibacillus boronitolerans is 96h, and the OD is about 2.5, the fermentation filtrate can effectively inhibit the growth of Magnaporthe oryzea, and the antagonistic efficiency reaches About 57.69%.
实施例4分离细菌发酵滤液粗提物的生防特性鉴定Example 4 Identification of Biocontrol Characteristics of Crude Extracts of Separated Bacterial Fermentation Filtrate
根据本申请的一个实施例，采用乙酸乙酯萃取法和硫酸铵沉淀提取法对具有生防效果的细菌发酵液进行粗提取，并采用打孔法对粗提物的抑菌活性进行鉴定。According to an embodiment of the present application, the ethyl acetate extraction method and the ammonium sulfate precipitation extraction method are used to crudely extract the bacterial fermentation broth with biocontrol effects, and the antibacterial activity of the crude extract is identified by the perforation method.
根据本申请的一个实施例，打孔法的具体操作步骤如下：According to an embodiment of the present application, the specific operation steps of the punching method are as follows:
1)配置PDA固体培养基，并将其冷却至55℃左右；或者将已凝固的PDA培养基加热融化并冷却至55℃左右；1) Prepare PDA solid medium and cool it to about 55°C; or heat and melt the solidified PDA medium and cool it to about 55°C;
2)待培养基冷却后，在培养基的中线部分，距中心点各3.5cm，用0.8cm的打孔器小心按压打孔，用灭菌后的镊子夹出多余的培养基，在平板中央接种1个供试病原菌菌饼(直径为8mm)；2) After the medium is cooled, in the midline part of the medium, 3.5cm from the center point, use a 0.8cm puncher to carefully press and punch holes, and use the sterilized tweezers to grab the excess medium and place it in the center of the plate. Inoculate 1 test pathogenic bacteria cake (diameter 8mm);
3)往打好的孔里小心注入提取物。本实验左边孔里为对照溶剂，右边为提取物。3) Carefully inject the extract into the punched hole. In this experiment, the left hole is the control solvent, and the right is the extract.
根据本申请的一个实施例，制备粗提物的具体操作如下：According to an embodiment of the present application, the specific operation for preparing the crude extract is as follows:
(1)乙酸乙酯萃取法制备粗提物(1) Preparation of crude extract by ethyl acetate extraction method
将耐硼赖氨酸芽孢杆菌于37℃，以150r/min转速培养，然后转接菌液，以200ml菌液/500ml LB培养基的装液量，至终浓度为0.02OD 600/ml，继续150r/min转速，于37℃培养至96h，使得OD值达到2.5左右，收集发酵产物，8000r/min离心去除沉淀，获得发酵上清液，然后用0.22μm的滤膜过滤，即得到无菌的发酵滤液。取400ml无菌发酵滤液，加入等体积的乙酸乙酯溶剂，振荡萃取4次，萃取结束后，使用分液漏斗将上层的乙酸乙酯全部取出，获得萃取液。合并4次萃取液，加入500ml旋蒸瓶中进行旋转蒸发浓缩，至乙酸乙酯完全蒸干，然后加入丙酮溶解残留浸膏，使其浓度为1mg/ml、5mg/ml、10mg/ml、15mg/ml，分别测试粗提物的抑菌活性。对照溶剂为丙酮。图3为根据本申请的一个实施例，采用乙酸乙酯萃取法制备耐硼赖氨酸芽孢杆菌发酵96h的发酵液粗提物，检测其对稻瘟菌的生长的抑制效果。如图3所示的实验结果，不同浓度的粗提物均对稻瘟病菌有不同程度的抑制作用。 Incubate boron-resistant lysine bacillus at 37°C at a speed of 150r/min, then transfer the bacterial solution to 200ml bacterial solution/500ml LB medium to fill the volume to a final concentration of 0.02OD 600 /ml, continue Cultivate at 150r/min for 96h at 37℃ to make the OD value reach about 2.5. Collect the fermentation product, centrifuge at 8000r/min to remove the precipitate to obtain the fermentation supernatant, and then filter it with a 0.22μm filter membrane to obtain a sterile Fermentation filtrate. Take 400 ml of the aseptic fermentation filtrate, add an equal volume of ethyl acetate solvent, shake and extract 4 times, after the extraction, use a separatory funnel to take out all the ethyl acetate in the upper layer to obtain the extract. Combine the four extracts and add them to a 500ml rotary evaporation flask for rotary evaporation concentration until the ethyl acetate is completely evaporated to dryness, and then add acetone to dissolve the remaining extract to make the concentration 1mg/ml, 5mg/ml, 10mg/ml, 15mg /ml, respectively test the antibacterial activity of the crude extract. The control solvent is acetone. Fig. 3 shows a 96-hour fermentation broth crude extract of boron-lysine-resistant Bacillus fermented by an ethyl acetate extraction method prepared according to an embodiment of the present application, and its inhibitory effect on the growth of Pyricularia oryzae is tested. As shown in the experimental results shown in Figure 3, the crude extracts of different concentrations have different degrees of inhibition on the rice blast fungus.
(2)硫酸铵沉淀法制备粗提物(2) Preparation of crude extract by ammonium sulfate precipitation method
首先，确定最佳硫酸铵浓度。获得耐硼赖氨酸芽孢杆菌在96h最佳发酵条件下的大量发酵液，收集发酵液，4℃、8000rpm离心10min去除菌体。取上清液各200ml，分别缓慢加入硫酸铵至饱和度为10％、20％、30％、40％、50％、60％、70％或80％，4℃过夜沉淀，8000rpm离心10min收集沉淀，沉淀用PBS(pH7.0)溶解并定容至相同体积，测定其抑菌活性，每个处理重复3次，活性最强的组分的硫酸铵饱和度即为制备抗菌粗提物所需的硫酸铵最适饱和度。First, determine the optimal ammonium sulfate concentration. Obtain a large amount of fermentation broth of boron-resistant lysine bacillus under the optimal fermentation conditions for 96 hours, collect the fermentation broth, and centrifuge at 4°C and 8000 rpm for 10 minutes to remove the bacteria. Take 200ml each of the supernatants, slowly add ammonium sulfate to the saturation of 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, precipitate overnight at 4°C, and collect the precipitate by centrifugation at 8000 rpm for 10 minutes , The precipitate is dissolved in PBS (pH 7.0) and dilute to the same volume to determine its antibacterial activity. Each treatment is repeated 3 times. The ammonium sulfate saturation of the most active component is required for the preparation of the crude antibacterial extract. The optimum saturation of ammonium sulfate.
接着，采用硫酸铵沉淀法制备粗提物。取400ml耐硼赖氨酸芽孢杆菌在96h最佳发酵条件下的发酵上清液，边搅拌边向上清液中加入硫酸铵，使其饱和度达到50％、60％、70％或80％，4℃过夜沉淀。8000rpm离心10min收集沉淀，沉淀用PBS(pH7.0)溶解并定容至10ml，测定其抑菌活性，对照溶剂为PBS(pH7.0)。图4为根据本申请的一个实施例，采用硫酸铵沉淀法制备耐硼赖氨酸芽孢杆菌发酵96h的发酵液粗提物，检测其对稻瘟菌的生长的抑制效果。如图4所示可知，不同硫酸铵饱和度沉淀实验中，硫酸铵饱和度为80％的发酵液粗提物对稻瘟菌的生长的抑制效果最为明显。Next, the crude extract was prepared by the ammonium sulfate precipitation method. Take 400ml of the fermentation supernatant of Boron-Lysine-resistant Bacillus under the optimal fermentation conditions for 96 hours, and add ammonium sulfate to the supernatant while stirring to make the saturation reach 50%, 60%, 70% or 80%. Precipitation overnight at 4°C. The precipitate was collected by centrifugation at 8000 rpm for 10 min. The precipitate was dissolved in PBS (pH 7.0) and the volume was adjusted to 10 ml to determine its antibacterial activity. The control solvent was PBS (pH 7.0). Fig. 4 shows a 96-hour fermentation broth crude extract of boron-lysine-resistant Bacillus fermented by an ammonium sulfate precipitation method prepared according to an embodiment of the present application, and its inhibitory effect on the growth of Pyricularia oryzae is tested. As shown in Figure 4, in the precipitation experiments with different ammonium sulfate saturation degrees, the crude extract of the fermentation broth with 80% ammonium sulfate saturation has the most obvious inhibitory effect on the growth of Pyricularia oryzae.
根据本申请的一个实施例，可以通过将生防细菌的无菌发酵滤液或其粗提物(例如本申请的耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)发酵滤液或其粗提物)与介质混合，以抑制致病菌生长，进而预防植物感染致病菌或者治疗已感染致病菌的植物。根据本申请的一个实施例，介质为任何可以携带致病菌的载体，例如介质可以包括水稻的种子、植物本身(即水稻生长的各个时期的苗)，也可以包括水稻生长的土壤、水环境，甚至培养植物的培养基等中的一者或者多者。根据本申请的一个实施例，致病菌可以是稻瘟病菌(Magnaporthe oryzea)。当然，不应当限定致病菌的种类，本申请的耐硼赖氨 酸芽孢杆菌(Lysinibacillus boronitolerans)可以一定程度上抑制其生长的细菌、真菌等都应视为本申请所说的致病菌。According to an embodiment of the present application, the sterile fermentation filtrate or crude extract of biocontrol bacteria (for example, the fermentation filtrate or crude extract of Lysinibacillus boronitolerans of the present application) can be combined with the medium Mix to inhibit the growth of pathogenic bacteria, thereby preventing plants from infecting pathogenic bacteria or treating plants that have been infected with pathogenic bacteria. According to an embodiment of the present application, the medium is any carrier that can carry pathogenic bacteria. For example, the medium may include rice seeds, the plant itself (that is, seedlings at various stages of rice growth), and may also include the soil and water environment where the rice grows. , Even one or more of the medium for cultivating plants. According to an embodiment of the present application, the pathogenic bacteria may be Magnaporthe oryzea. Of course, the types of pathogenic bacteria should not be limited. The bacteria and fungi that can inhibit the growth of Lysinibacillus boronitolerans in this application to a certain extent should be regarded as pathogenic bacteria mentioned in this application.
根据本实施例及图3和图4显示的实验结果可知，耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)发酵滤液中多种成分均对稻瘟病菌(Magnaporthe oryzea)具有不同程度的抑制作用。所以，可以将耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)作为底盘微生物底盘细胞，用于大量生产具有抑菌活性的发酵滤液，从而满足农业及生产生活上对杀菌剂的需求。根据本申请的一个实施例，还可以将耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)为基础进一步改进，将其改进为可以大量生产某一种或者多种具有特定抑菌活性的蛋白或者其他物质的改造模块，进而增强其制备的抑制稻瘟病菌(Magnaporthe oryzea)生长的杀菌剂的功能，提高其制备抑制稻瘟病菌(Magnaporthe oryzea)生长的杀菌剂的效率。According to the present example and the experimental results shown in Figures 3 and 4, it can be seen that various components in the fermentation filtrate of Lysinibacillus boronitolerans have different degrees of inhibition on Magnaporthe oryzea. Therefore, Lysinibacillus boronitolerans can be used as chassis microbial chassis cells for mass production of fermentation filtrate with antibacterial activity, so as to meet the demand for fungicides in agriculture, production and life. According to an embodiment of the present application, it can be further improved based on Lysinibacillus boronitolerans, which can be used to mass-produce one or more proteins or other substances with specific antibacterial activity The modification module further enhances the function of the fungicide for inhibiting the growth of Magnaporthe oryzea, and improves the efficiency of preparing the fungicide for inhibiting the growth of Magnaporthe oryzea.
根据本申请的一个实施例，还可以将耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)制作成杀菌剂，用以抑制稻瘟病菌(Magnaporthe oryzea)等致病菌的生长。根据本申请的一个实施例，杀菌剂包括耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)发酵滤液或其粗提物以及辅料。根据本申请的一个实施例，杀菌剂包括以耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)为基础的细胞改造模块制备的发酵液以及辅料。根据本申请的一个实施例，杀菌剂中耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)发酵液的发酵时长为96h，粗提物为采用乙酸乙酯萃取法和硫酸铵沉淀法获得的提取物。根据本申请的一个实施例，改造模块为可以大量生产某一种或者多种具有特定抑菌活性的蛋白或者其他物质的改造模块。根据本申请的一个实施例，辅料为水、液体培养基、固体培养基、甘油中的一种或者多种。根据本申请的一个实施例，根据可以杀菌剂的贮藏条件调节甘油的比例，例如甘油的体积比可以为10％、25％、50％、75％等。According to an embodiment of the present application, Lysinibacillus boronitolerans can also be made into a fungicide to inhibit the growth of pathogenic bacteria such as Magnaporthe oryzea. According to an embodiment of the present application, the bactericide includes the fermentation filtrate of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) or its crude extract and auxiliary materials. According to an embodiment of the present application, the bactericide includes a fermentation broth prepared by a cell modification module based on Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) and auxiliary materials. According to an embodiment of the present application, the fermentation time of the fermentation broth of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) in the fungicide is 96 hours, and the crude extract is an extract obtained by an ethyl acetate extraction method and an ammonium sulfate precipitation method. According to an embodiment of the present application, the modification module is a modification module that can mass-produce one or more proteins or other substances with specific antibacterial activity. According to an embodiment of the present application, the auxiliary material is one or more of water, liquid culture medium, solid culture medium, and glycerol. According to an embodiment of the present application, the proportion of glycerin can be adjusted according to the storage conditions of the bactericide. For example, the volume ratio of glycerin can be 10%, 25%, 50%, 75%, etc.
根据本申请的一个实施例，还可以将本申请的耐硼赖氨酸芽孢杆菌与其他具有生物防治功能的细菌、真菌混合，制成对多种致病菌具有良好抑制作用的制剂。According to an embodiment of the present application, the boron-lysine-resistant bacillus of the present application can also be mixed with other bacteria and fungi with biological control functions to prepare a preparation that has a good inhibitory effect on a variety of pathogenic bacteria.
根据本申请的一个实施例，预防或者治疗致病菌感染植物的方法，包括将前文所述的含有耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)发酵液或其粗提物的杀菌剂，将其与介质混合，进而抑制介质表面致病菌的生长。根据本申请的一个实施例，也可以直接将耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)培养并调配至所需浓度后，将其与介质混合。根据本申请的一个实施例，介质为任何可以携带致病菌的载体，例如介质可以包括水稻的种子、植物本身(即水稻生长的各个时期的苗)，也可以包括水稻生长的土壤、水环境，甚至培养植物的培养基等中的一者或者多者。根据本申请的一个实施例，致病菌可以是稻瘟病菌(Magnaporthe oryzea)。当然，不应当限定致病菌的种类，本申请的耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)，以及与其混合的其他生防菌可以一定程度上抑制其生长的细菌、真菌等都应视为本申请所说的致病菌。According to an embodiment of the present application, the method for preventing or treating pathogenic bacteria infecting plants includes applying the aforementioned fungicide containing the fermentation broth or crude extract of Lysinibacillus boronitolerans to Mix with the medium to inhibit the growth of pathogenic bacteria on the surface of the medium. According to an embodiment of the present application, it is also possible to directly cultivate and mix Lysinibacillus boronitolerans to a desired concentration, and then mix it with the medium. According to an embodiment of the present application, the medium is any carrier that can carry pathogenic bacteria. For example, the medium may include rice seeds, the plant itself (that is, seedlings at various stages of rice growth), and may also include the soil and water environment where the rice grows. , Even one or more of the medium for cultivating plants. According to an embodiment of the present application, the pathogenic bacteria may be Magnaporthe oryzea. Of course, the types of pathogenic bacteria should not be limited. The Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) of this application, as well as other biocontrol bacteria mixed with it, which can inhibit their growth to a certain extent, should be regarded as bacteria and fungi. The pathogenic bacteria mentioned in this application.
稻瘟病是水稻的重要病害，给农业生产造成了严重的经济损失。由于大量使用化学农药给环境和食品也带来了巨大的威胁。耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)不仅可以很大程度上降低稻瘟病的发生，而且于生态环境无任何威胁。进一步地，如可以通过对生防菌代谢物合成元件、装置的基础上，组装途径、合成模块，并模块或***进行的理性修饰，从而产生更强效抑菌活性；或通过揭示基因线路和调控网络，合理设计或优化逻辑基因线路和功能基因线路，使之成为最小细胞工厂从而产生强效抑菌活性，则可以为稻瘟病的防治带来新的方向。Rice blast is an important disease of rice, causing serious economic losses to agricultural production. The extensive use of chemical pesticides also poses a huge threat to the environment and food. Boron-resistant lysine bacillus (Lysinibacillus boronitolerans) can not only reduce the occurrence of rice blast to a large extent, but also has no threat to the ecological environment. Further, for example, it is possible to assemble pathways, synthetic modules, and rationally modify modules or systems on the basis of synthetic elements and devices for biocontrol bacteria metabolites to produce more potent antibacterial activity; or by revealing gene circuits and Regulating the network, rationally designing or optimizing the logical gene circuit and functional gene circuit, making it the smallest cell factory to produce potent antibacterial activity, can bring a new direction for the prevention and treatment of rice blast.
上述实施例仅供说明本发明之用，而并非是对本发明的限制，有关技术领 域的普通技术人员，在不脱离本发明范围的情况下，还可以做出各种变化和变型，因此，所有等同的技术方案也应属于本发明公开的范畴。The above-mentioned embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. Those of ordinary skill in the relevant technical fields can also make various changes and modifications without departing from the scope of the present invention. Therefore, all The equivalent technical solutions should also belong to the scope of the disclosure of the present invention.

Claims (12)

  1. 一种耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)，于2019年10月8日保藏于中国典型培养物保藏中心，简称CCTCC，保藏编号为CCTCC NO.M 2019773。A boron-resistant lysine bacillus (Lysinibacillus boronitolerans) was deposited in the China Center for Type Culture Collection on October 8, 2019, referred to as CCTCC, and the deposit number is CCTCC NO.M 2019773.
  2. 如权利要求1所述的耐硼赖氨酸芽孢杆菌发酵液在抑制稻瘟病菌(Magnaporthe oryzea)生长方面的应用。The use of the boron-resistant lysine bacillus fermentation broth of claim 1 in inhibiting the growth of Magnaporthe oryzea.
  3. 一种杀菌剂，包括：如权利要求1所述的耐硼赖氨酸芽孢杆菌的发酵液以及辅料。A bactericide, comprising: the fermentation broth of boron-lysine-resistant Bacillus spp according to claim 1 and auxiliary materials.
  4. 根据权利要求3所述的杀菌剂，所述辅料为水、液体培养基、固体培养基、甘油中的一种或者多种。The bactericide according to claim 3, wherein the auxiliary material is one or more of water, liquid culture medium, solid culture medium, and glycerin.
  5. 根据权利要求3所述的杀菌剂，其中所述的耐硼赖氨酸芽孢杆菌的发酵液为如权利要求1所述的硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans)经24-96小时培养，OD 600为4.8-2.5时的发酵液。 The fungicide according to claim 3, wherein the fermentation broth of Boron-Lysine-resistant Bacillus is the Lysinibacillus boronitolerans according to claim 1 after 24-96 hours of culture, OD 600 is the fermentation broth at 4.8-2.5 hours.
  6. 如权利要求1所述的耐硼赖氨酸芽孢杆菌在制备抑制稻瘟病菌(Magnaporthe oryzea)生长的杀菌剂中的应用。The use of the boron-lysine-resistant bacillus according to claim 1 in the preparation of a fungicide for inhibiting the growth of Magnaporthe oryzea.
  7. 如权利要求1所述的耐硼赖氨酸芽孢杆菌作为底盘细胞在制备抑制稻瘟病杀菌剂中的应用。The use of the boron-resistant lysine-resistant Bacillus as claimed in claim 1 as a bottom plate cell in the preparation of a fungicide for inhibiting rice blast.
  8. 一种如权利要求1所述的耐硼赖氨酸芽孢杆菌作为底盘细胞为基础的改造模块。A Bacillus resistant to boron-lysine as claimed in claim 1 is used as a chassis cell-based transformation module.
  9. 如权利要求8所述的改造模块在制备抑制稻瘟病菌(Magnaporthe oryzea)生长的杀菌剂中的应用。The application of the modified module according to claim 8 in the preparation of a fungicide for inhibiting the growth of Magnaporthe oryzea.
  10. 一种用于防治稻瘟病的方法，包括：A method for controlling rice blast, including:
    获取如权利要求3所述的杀菌剂；以及Obtain the fungicide according to claim 3; and
    将所述杀菌剂与介质混合。The bactericide is mixed with the medium.
  11. 根据权利要求10所述的方法，其中，所述介质为携带稻瘟病菌(Magnaporthe oryzea)的载体。The method according to claim 10, wherein the medium is a vector carrying Magnaporthe oryzea.
  12. 根据权利要求11所述的方法，其中，所述载体为种子、植物、植物生长的土壤、培养植物的培养基中的一者或多者。The method according to claim 11, wherein the carrier is one or more of a seed, a plant, the soil in which the plant grows, and a medium for cultivating the plant.
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